JP2022062157A - Site-specific antibody-drug conjugation through glycoengineering - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide binding polypeptides (e.g., antibodies), and effector moiety conjugates thereof (e.g., antibody-drug conjugates or ADCs), comprising a site-specifically engineered drug-glycan linkage within native or engineered glycans of the binding polypeptide.

SOLUTION: A binding polypeptide comprises at least one modified glycan comprising at least one moiety of a formula (IV): -Gal-Sia-C(H)=N-Q-CON-X, where: A) Q is NH or O; B) CON is a connector moiety; C) X is an effector moiety; D) Gal is a constituent derived from galactose; and E) Sia is a constituent derived from a sialic acid, where Sia is present or absent.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

This application is filed in US Provisional Patent Application No. 61 / 767,724 entitled "Site-Specific Antibody Drug Conjugation by Sugar Engineering" filed on March 11, 2013; "High Glycosylation" filed on March 11, 2013. US Provisional Patent Application No. 61 / 767,710 entitled "Chemical Binding Polypeptides" and "Fc-Containing Polypeptides with Modified Glycosylation and Reduced Effector Function" filed March 11, 2013. It claims the priority of the title US Provisional Patent Application No. 61 / 767,715. The contents of the aforementioned application are incorporated herein by reference in their entirety.

Treatment of cancer remains a significant challenge for humankind. Current standard treatments have saved the lives of many patients, including surgery, irradiation, and chemotherapy, but more effective treatments, especially more effective and large targets in the treatment window. There is a great need for specific treatments. One of these target-specific therapies uses antibody-drug conjugates (ADCs), where antigen-specific antibodies target non-specific chemotherapeutic agents to the tumor site. do. These molecules have been shown to have efficacy and a good safety profile in clinical settings. However, the development of such therapies can be difficult due to many factors, including the antibody itself and the stability of ligation, which can have a significant impact on tumor specificity, thus reducing its effectiveness. .. ADCs have high non-specific binding and low circulation stability, so they are removed by normal tissue before reaching the tumor. In addition, ADCs with significant subpopulations with high drug loading can produce aggregates that are eliminated by macrophages, thus reducing their half-life. Therefore, there is an increasing need for decisive process control and improvement, as well as prevention of complications such as product aggregation and non-specific toxicity from IgG.

Although ADCs produced by current methods are effective, the development of such treatments can be difficult due to the often heterogeneous mixtures resulting from the chemical reactions of the conjugations used. For example, drug conjugation to antibody lysine residues is complicated by the fact that there are many lysine residues (approximately 30) in the antibody available for conjugation. Since the optimal number of drug-to-antibody ratios (DARs) is very low (eg, approximately 4: 1), lysine conjugation often produces a very heterogeneous profile. In addition, many lysines are located at the critical antigen binding sites of the CDR regions and drug conjugation can result in reduced antibody affinity. Thiol-mediated conjugation, on the other hand, primarily targets the eight cysteines involved in the disulfide bond of the hinge, but in various preparations, four of the eight cysteines are consistent. It is still difficult to predict and identify whether to conjugate. More recently, genetic manipulation of free cysteine residues has enabled site-specific conjugation in thiol-based chemistries, but such linkages often exhibit highly variable stability. , Drug-linker undergoes an exchange reaction with albumin and other thiol-containing serum molecules. Therefore, site-specific conjugation strategies that produce ADCs with defined conjugation sites and stable linkages ensure drug conjugation and minimize adverse effects on antibody structure or function. Extremely useful.

The present disclosure provides a binding polypeptide (eg, an antibody) and a conjugate thereof (eg, a drug conjugate) of the effector moiety. In certain embodiments, the conjugate comprises a site-specifically modified drug-glycan linkage within the natural or modified glycan of the binding polypeptide. The present disclosure also provides nucleic acids encoding antigen-binding polypeptides, recombinant expression vectors, and host cells for making such antigen-binding polypeptides. Also provided are methods of treating a disease with the antigen-binding polypeptides disclosed herein.

In certain embodiments, the binding polypeptide of the invention can be obtained by coupling effector moieties (eg, drug moieties) with stable (eg, oxime) linkages. This strategy provides a highly defined product with enhanced in vivo stability and reduced aggregation. In other embodiments, the conjugate of the effector moiety (eg, drug conjugate) is a terminal sugar residue of an IgG glycan (eg, terminal sialic acid or galactose residue) to provide additional site selectivity and uniformity. ) Can be formed by coupling to. Terminal sugar residues can be readily converted to reactive aldehyde forms by mild oxidation (eg, with sodium periodate). Oxidized sugar residues can then be conjugated to an aldehyde-reactive aminooxy drug-linker to provide a stable and uniform population of protein-drug conjugates (eg, ADCs).

Therefore, in one embodiment, the present invention has at least one formula (IV) :.
-Gal-Sia-C (H) = NQ-CON-X
Equation (IV)
[During the ceremony,
A) Q is NH or O,
B) CON is a connection part,
C) X is an effector moiety (eg, drug moiety or targeting moiety).
D) Gal is a component derived from galactose and is a component.
E) Sia is a component derived from sialic acid and is a component.
Sia is present or non-existent]
A binding polypeptide comprising at least one modified glycan comprising a moiety is provided.

In one embodiment, the modified glycan is a bifurcated glycan. In another embodiment, the bifurcated glycan is fucosylated or non-fucosylated. In another embodiment, the modified glycan comprises at least two parts of formula (IV) and Sia is present in only one of the two parts. In another embodiment, the modified glycan comprises at least two parts of formula (IV) and Sia is present in both parts. In another embodiment, the modified glycan is N-linked to the binding polypeptide.

In another embodiment, the binding polypeptide comprises an Fc domain. In another embodiment, the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid position 297 of the Fc domain by EU numbering. In another embodiment, the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid 298 of the Fc domain by EU numbering. In another embodiment, the Fc domain is human.

In another embodiment, the binding polypeptide comprises a CH1 domain. In one embodiment, the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid 114 of the CH1 domain by Kabat numbering. In one embodiment, the binding polypeptide is an antibody or immunoadhesin.

In one embodiment, the effector moiety is cytotoxic. In another embodiment, the cytotoxicity is selected from the group consisting of the cytotoxicities listed in Table 1. In another embodiment, the effector portion is a detector. In certain embodiments, the effector portion is a targeted portion. In one embodiment, the targeting moiety is a carbohydrate or glycopeptide. In another embodiment, the targeting moiety is a glycan.

In another embodiment, the connecting moiety comprises a pH sensitive linker, a disulfide linker, an enzyme sensitive linker, or another cleavable linker moiety. In another embodiment, the connecting moiety comprises a linker moiety selected from the group of linker moieties shown in Table 2 or 14.

In another aspect, the invention provides a composition comprising the binding polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. In one embodiment, the ratio of therapeutic or diagnostic effector moieties to the binding polypeptide is less than 4. In another embodiment, the ratio of therapeutic or diagnostic effector moieties to the binding polypeptide is about 2.

In another aspect, the invention provides a method of treating a patient in need thereof, comprising administering an effective amount of the composition of the invention.

In another embodiment, the invention provides an isolated polynucleotide encoding the binding polypeptide of the invention. In another aspect, the invention provides a vector comprising a polynucleotide. In another embodiment, the invention provides a host cell containing a polynucleotide or vector.

In yet another embodiment, the invention is a method of making the binding polypeptide of the invention, according to formula (I) :.
NH ₂ -Q-CON-X
Equation (I)
[During the ceremony,
A) Q is NH or O,
B) CON is a connection part,
C) X is the effector part]
Provided are methods comprising reacting an effector moiety of the same with a modified binding polypeptide containing an oxidized glycan.

In one embodiment, the modified binding polypeptide comprises an oxidized glycan produced by reacting the binding polypeptide containing the glycan with a mild oxidizing agent. In certain embodiments, the mild oxidant is sodium periodate. In certain embodiments, less than 1 mM sodium periodate is used. In one embodiment, the oxidizing agent is galactose oxidase. In another embodiment, the glycan-containing binding polypeptide comprises one or two terminal sialic acid residues. In another embodiment, the terminal sialic acid residue is introduced by treating the binding polypeptide with sialyltransferase, or a combination of sialyltransferase and galactosyltransferase.

It is a schematic diagram showing the synthesis of an antibody drug conjugate in which the toxin moiety is linked to the oxidized sialic acid residue of the glycan of the antibody using an oxime bond. FIG. 5 shows a Coomassie blue stained gel showing the expression and purification of glycosylation variants. It is a figure which shows the result of the surface plasmon resonance experiment which used to evaluate the binding to the recombinant human FcγRIIIa (V158 and F158) of the mutant of αβTCR HEBE1 IgG antibody. It is a figure which shows the result of the surface plasmon resonance experiment which used to evaluate the binding to recombinant human FcγRI of the mutant of αβTCR HEBE1 IgG antibody. It is a figure which shows the profile of the cytokine release from PBMC to TNFa, GM-CSF, IFNy, and IL10 in the presence of a mutant anti-αβTCR antibody (day 2). It is a figure which shows the profile of the cytokine release from PBMC to IL6, IL4, and IL2 in the presence of a mutant anti-αβTCR antibody (day 2). It is a figure which shows the profile of the cytokine release from PBMC to TNFa, GM-CSF, IFNy, and IL10 in the presence of a mutant anti-αβTCR antibody (day 4). It is a figure which shows the profile of the cytokine release from PBMC to IL6, IL4, and IL2 in the presence of a mutant anti-αβTCR antibody (day 4). It is a figure which shows the result of the experiment which investigated the expression level of 2C3 mutant by Western blot and surface plasmon resonance. It is a figure which shows the result of the experiment which investigated the glycosylation of the 2C3 mutant before and after the PNGase F treatment. It is a figure which shows the result of the SDS-PAGE experiment which investigates the glycosylation site for the 2C3 mutant isolated from the cell culture. It is a figure which shows the result of the surface plasmon resonance experiment which used to evaluate the binding of modified anti-CD52 to recombinant human FcγRIIIa (V158). The effector function of the modified molecule was evaluated using anti-CD52 containing the S298N / Y300S mutation in the Fc domain. Binding to CD52 peptide (A), binding to FcγRIIIa (V158, B), and control binding to mouse FcRn (C). It is a figure which shows the result of the surface plasmon resonance experiment which investigated the Fc binding property of a 2C3 mutant. FIG. 5 shows the results of a surface plasmon resonance experiment investigating the binding of modified anti-CD52 to both FcγRIIIa (Val158) (as described above) and FcγRIIIa (Phe158). Anti-CD52 antibodies containing the S298N / Y300S mutation in the Fc domain were used to evaluate the effector function of binding of modified molecules to FcγRIIIa (Val158, FIG. 14A) and FcγRIIIa (Phe58, FIG. 14B). It is a figure which shows the result of the analysis of C1q binding in S298N / Y300S mutant and WT2C3 control (A), and the Eliza analysis which confirms the equivalent coating of a well. It is a figure which shows the result of the plasmon resonance experiment which measures the binding kinetics of a 2C3 mutant to CD-52 peptide 741. It is a figure which shows the result of the plasmon resonance experiment which compares the antigen-binding affinity of WT anti-CD-52 2C3 and A114N hyperglycosylation mutant. It is a figure which shows the result of the charge characterization experiment by the isoelectric focusing and mass spectrometry for determining the glycan content of a 2C3 mutant. Continuation of FIG. 18-1. Continuation of Figure 18-2. FIG. 5 shows the results of concentration (Octet) and plasmon resonance experiments comparing antigen binding affinities of WT anti-CD-52 2C3 and mutants. It is a figure which shows the result of the SDS-PAGE experiment which demonstrates the further glycosylation of an anti-TEM1 A114N mutant. It is a figure which shows the result of SDS-PAGE and hydrophobic interaction chromatography analysis of A114N anti-Her2 mutant. It is a figure which shows the result of the SDS-PAGE experiment which demonstrates the conjugation of PEG to the 2C3 A114N mutant by aminooxy ligation. It is a figure which shows the result of the LC-MS experiment for determining the glycan content of the anti-TEM1 A114N hyperglycosylated mutant. FIG. 6 shows the results of LC-MS experiments to determine the glycan content of wild-type HER2 antibody and A114N anti-Her2 hyperglycosylated mutant. It is a figure which shows the exemplary method for performing the site-specific conjugation of an antibody by the method of this invention. It is a figure which shows the exemplary method for performing the site-specific conjugation of an antibody by the method of this invention. It is a figure which shows the exemplary method for performing the site-specific conjugation of an antibody by the method of this invention. It is a figure which shows the synthesis of aminooxy-Cys-MC-VC-PABC-MMAE and aminooxy-Cys-MC-VC-PABC-PEG8-Dol10 which are the exemplary effector part of this invention. It is a figure which shows the characterization information for a sialylated HER2 antibody. It is a figure which shows the characterization information for an oxidized sialylated anti-HER2 antibody. It is a figure which shows the hydrophobic interaction chromatograph with two different aminooxy groups of a complex carbohydrate (glycoconjugate) prepared with three different sialylated antibodies. It is a figure which shows the HIC chromatograph of the conjugate with AO-MMAE of the anti-Her2 A114 glycosylation mutant prepared using the GAM (+) chemical reaction. It is a figure which shows the comparison of the efficacy of anti-HER2 glycoconjugate and thiol conjugate in vitro. It is a figure which shows the comparison of the efficacy of the complex carbohydrate of anti-FAP B11 and the in vitro of a thiol conjugate. It is a figure which shows the comparison of the efficacy of the anti-HER2 glycoconjugate and the thiol conjugate in vivo in the Her2 + tumor cell xenograft model. It is a figure which shows the result of the LC-MS experiment for determining the glycan content of the anti-αβTCR antibody of the mutant containing the S298N / Y300S mutation. It is a figure which shows the result of the circular dichroism experiment for determining the relative thermal stability of the wild type anti-αβTCR antibody, and the mutant anti-αβTCR antibody containing the S298N / Y300S mutation. It is a figure which shows the result of the cell proliferation assay against the ADC prepared with the anti-HER antibody carrying A114N hyperglycosylation mutation and AO-MMAE.

The present disclosure provides a binding polypeptide (eg, an antibody) and a conjugate thereof (eg, a drug conjugate) of the effector moiety. In certain embodiments, the conjugate comprises a site-specifically modified drug-glycan linkage within a natural or modified glycan of an antigen-binding polypeptide such as an IgG molecule. The present disclosure also provides nucleic acids encoding antigen-binding polypeptides, recombinant expression vectors, and host cells for making such antigen-binding polypeptides. Also provided are methods of treating a disease with the antigen-binding polypeptides disclosed herein.

I. Definitions As used herein, the term "binding polypeptide" or "binding polypeptide" is a polypeptide comprising at least one binding site responsible for selective binding to a target antigen of interest (eg, a human antigen). Means (eg, antibody). Exemplary binding sites include antibody variable domains, ligand binding sites of receptors, or receptor binding sites of ligands. In some embodiments, the binding polypeptide of the invention comprises a plurality of (eg, two, three, four, or more) binding sites.

As used herein, the term "natural residue" is naturally occurring at a particular amino acid position of a binding polypeptide (eg, an antibody or fragment thereof) and has been modified, introduced or modified by humans. Means no amino acid residues. As used herein, the term "modified binding polypeptide" or "modified binding polypeptide" is a binding polypeptide comprising at least one unnaturally mutated amino acid residue (eg, for example. Contains antibodies or fragments thereof).

As used herein, the term "specifically binds" refers to an antibody or antigen-binding fragment thereof having a maximum of about 1 x ^10-6 M, 1 x ^10-7 M, 1 x ^10-8 M, and the like. Ability to bind antigen with a dissociation constant (Kd) of 1 × 10 ^-9 M, 1 × 10 ^-10 M, 1 × 10 ^-11 M, 1 × 10 ^-12 M, or less, and / or non-specific It means the ability to bind to an antigen with an affinity that is at least twice as high as that of an antigen.

As used herein, the term "antibody" means an aggregate (eg, an intact antibody molecule, antibody fragment, or variant thereof) having a significant known specific immune response activity against an antigen of interest. do. Antibodies and immunoglobulins include light and heavy chains with or without interchain covalent bonds. The basic immunoglobulin structure of the vertebrate system is relatively well understood.

As discussed in more detail below, the generic term "antibody" includes five distinct classes of antibodies that can be biochemically distinguished. Although all five classes of antibodies are expressly within the scope of this disclosure, the discussion below is largely for IgG class immunoglobulin molecules. With respect to IgG, an immunoglobulin comprises two identical light chains having a molecular weight of approximately 23000 daltons and two identical heavy chains having a molecular weight of 53000-70000. The four chains are connected in a "Y" shape by disulfide bonds, and the light chain is connected to the heavy chain at the portion starting from the opening of "Y" and continuing to the variable region.

Immunoglobulin light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. In general, light and heavy chains are covalently linked to each other, and the location of the "tail" of the two heavy chains depends on whether the immunoglobulin is a hybridoma, a B cell, or a genetically engineered host cell. When produced, they are linked to each other by covalent disulfide linkage or non-covalent linkage. In heavy chains, the amino acid sequence is arranged from the N-terminus of the Y-shaped fork-like termination to the C-terminus of the bottom of each chain. For those skilled in the art, heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), in which some subclasses (eg, γ1 to γ4) are present. Let's understand that there is. It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Subclasses of immunoglobulin isotypes (eg, IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to provide functional specialization. Modified forms of each of these classes and isotypes can be readily recognized by those of skill in the art in view of the present disclosure and are also within the scope of the present disclosure.

Both light and heavy chains are divided into structurally homologous regions and functionally homologous regions. The term "region" means an element or portion of an immunoglobulin or antibody chain and includes a constant or variable region and a further distinct element or portion of said region. For example, the light chain variable regions include "complementarity determining regions" or "CDRs" interspersed within the "framework regions" or "FRs" defined herein.

Immunoglobulin heavy or light chain regions may be defined as "constant" (C) or "variable" (V) regions, where "constant regions" are within the regions of diverse class members. It is so defined based on the relative lack of changes in the array, or, in the case of "variable regions", the significant changes within the regions of the various class members. The terms "stationary region" and "variable region" may also be used functionally. In this regard, it will be appreciated that the variable region of the immunoglobulin or antibody determines the recognition and specificity of the antigen. Conversely, constant regions of immunoglobulins or antibodies confer important effector functions such as, for example, secretion, transplacental transfer, Fc receptor binding, complement binding. The subunit structure and three-dimensional arrangement of constant regions of various classes of immunoglobulins are well known.

The constant and variable regions of the immunoglobulin heavy and light chains are folded into domains. The term "domain" includes a beta pleated sheet and / or a peptide loop stabilized by interchain disulfide bonds and the like (eg, including 3 to 4 peptide loops), heavy or light chain spherical. Means an area. The constant region domains on the immunoglobulin light chain are referred to interchangeably as "light chain constant region domain", "CL region", or "CL domain", respectively. The constant domains on the heavy chain (eg, hinge, CH1, CH2, or CH3 domains) are referred to interchangeably as "heavy chain constant region domain", "CH" region domain, or "CH domain", respectively. The variable domains on the light chain are referred to interchangeably as "light chain variable domain", "VL domain", or "VL domain", respectively. The variable domains on the heavy chain are referred to as "heavy chain variable region domain", "VH region domain", or "VH domain" in the same meaning.

By convention, the numbering of variable constant region domains increases as the variable constant region domain becomes more distal from the antigen binding site or amino terminus of the immunoglobulin or antibody. The N-terminus of each of the heavy and light chains of the immunoglobulin is a variable region and the C-terminus is a constant region, but in reality the carboxy terminus of the heavy and light chains is contained in the CH3 and CL domains, respectively. Thus, the light chain domains of immunoglobulins are aligned in the VL-CL arrangement and the heavy chain domains are aligned in the VH-CH1-hinge-CH2-CH3 arrangement.

Amino acid positions in heavy chain constant regions such as amino acid positions in CH1, hinges, CH2, CH3, and CL domains may be numbered according to the Kabat index numbering system (Kabat et al., "Sequences of Products of Immunological Interest", United States Health. See Ministry of Health, 5th Edition, 1991). Alternatively, the amino acid positions of the antibody may be numbered according to the EU index numbering system (see Kabat et al., Ibid.).

As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain.

As used herein, the term "CH1 domain" refers to, for example, the first (at the very end of the amino terminus) of an immunoglobulin heavy chain spanning about 114-223 positions (EU 118-215 positions) of Kabat's numbering system. Includes constant region domain. The CH1 domain is in contact with the VH domain of the immunoglobulin heavy chain and the amino terminus of the hinge region and does not form part of the Fc region.

As used herein, the term "hinge region" includes a portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three individual domains, the upper, central, and lower hinge domains (Roux et al., J. Immunol., 1998, 161), p. 4083).

As used herein, the term "CH2 domain" includes, for example, a portion of a heavy chain immunoglobulin molecule spanning positions 244 to 360 (EU231 to 340) in Kabat's numbering system. The CH2 domain is unique in that it does not tightly pair with other domains. In contrast, two N-linked branched carbohydrate chains intervene between the two CH2 domains of the intact native IgG molecule. In one embodiment, the binding polypeptide of the present disclosure comprises a CH2 domain derived from an IgG1 molecule (eg, a human IgG1 molecule).

As used herein, the term "CH3 domain" refers to a heavy chain immunoglobulin molecule that spans approximately 110 residues from the N-terminus of the CH2 domain, eg, at positions 361-476 (EU341-445) of Kabat's numbering system. Including a part. The CH3 domain typically forms the C-terminal portion of the antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (eg, the CH4 domain in the μ chain of IgM and the e chain of IgE). In one embodiment, the binding polypeptide of the present disclosure comprises a CH3 domain derived from an IgG1 molecule (eg, a human IgG1 molecule).

As used herein, the term "CL domain" includes, for example, the constant region domain of an immunoglobulin light chain ranging from about 107A to 216 of Kabat. The CL domain is adjacent to the VL domain. In one embodiment, the binding polypeptide of the present disclosure comprises a CL domain derived from a kappa light chain (eg, a human kappa light chain).

As used herein, the term "Fc region" begins in the hinge region just upstream of the papain cleavage site (ie, with the first residue of the heavy chain constant region as 114, IgG residue 216) and of the antibody. It is defined as the heavy chain constant region portion ending at the C-terminus. Therefore, the complete Fc region includes at least the hinge domain, CH2 domain, and CH3 domain.

As used herein, the term "natural Fc", whether in monomeric or multimeric form, is a non-antigen binding obtained by digestion of an antibody or generated by other means. It means a molecule containing a sequence of sex fragments and may include a hinge region. The origin of the original immunoglobulin of the native Fc is preferably human origin and may be any immunoglobulin, but IgG1 and IgG2 are preferred. Natural Fc molecules are composed of monomeric polypeptides that can be linked in the form of dimers or multimers by covalent (ie, disulfide bond) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a natural Fc molecule depends on the class (eg, IgG, IgA, and IgE) or subclass (eg, IgG1, IgG2, IgG3, IgA1, and IgGA2). It ranges from 1 to 4. An example of a native Fc is a disulfide-bonded dimer obtained by papain digestion of IgG. As used herein, the term "natural Fc" is a generic term for the forms of monomers, dimers, and multimers.

As used herein, the term "Fc variant" means a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Thus, the term "Fc variant" can include molecules or sequences humanized from non-human native Fc. In addition, the native Fc comprises a region that is not required for the antibody-like binding polypeptides of the invention and can be removed to provide structural features or biological activity. Thus, the term "Fc variant" refers to (1) formation of disulfide bonds, (2) incompatibility with selected host cells, and (3) N-terminal when expressed in selected host cells. Affects heterogeneity, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC). , Or a molecule or sequence in which one or more natural Fc sites or residues are deleted or modified with one or more Fc sites or residues involved.

As used herein, the term "Fc domain" includes native Fc and Fc variants, as well as the sequences defined above. Along with Fc variants and native Fc molecules, the term "Fc domain" includes molecules in the form of monomers or multimers, whether obtained from whole antibodies by digestion or by other means. ..

As pointed out above, the variable region of the antibody allows the antibody to selectively recognize and specifically bind to epitopes on the antigen. That is, the VL domain and VH domain of the antibody combine to form a variable region (Fv) that defines a three-dimensional antigen binding site. This quaternary structure of the antibody forms the antigen binding site present at the end of each arm of Y. More specifically, the antigen binding site is defined by three complementarity determining regions (CDRs) above each of the heavy and light chain variable regions. As used herein, the term "antigen binding site" includes a site that specifically binds to (and immunoreacts with) an antigen (eg, a cell surface or a soluble antigen). Antigen binding sites include the variable regions of the heavy and light chains of immunoglobulins, and the binding sites formed by these variable regions determine the specificity of the antibody. Antigen binding sites are formed by variable regions that vary from antibody to antibody. The modified antibody of the present disclosure comprises at least one antigen binding site.

In certain embodiments, the binding polypeptide of the present disclosure comprises at least two antigen binding domains that result in association of the binding polypeptide with the selected antigen. The antigen-binding domain does not necessarily have to be derived from the same immunoglobulin molecule. In this regard, the variable region may be of any type of animal that can initiate a humoral response and be induced to produce immunoglobulins against the desired antigen. Thus, the variable region of the binding polypeptide may be, for example, of mammalian origin, eg, human, mouse, rat, goat, sheep, non-human primate (eg, cynomolgus monkey, macaque, etc.), wolf, or camel. It may be (eg, from camels, llamas, and related species).

In naturally occurring antibodies, the six CDRs present on each monomeric antibody are specifically arranged to form an antigen binding site when the antibody exhibits its three-dimensional structure in an aqueous environment. It is a short non-adjacent sequence of amino acids. The rest of the heavy and light chain variable regions show little intermolecular variability in the amino acid sequence and are called framework regions. The framework region generally takes a β-sheet configuration and the CDRs connect to the β-sheet structure or possibly form loops that form part of the β-sheet structure. Thus, these framework regions act to form a skeleton that places the six CDRs in the correct position through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDR defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to immunoreactive antigen epitopes.

Exemplary binding polypeptides of the invention include antibody variants. As used herein, the term "antibody variant" includes an antibody in a synthesized, modified form that has been modified to be non-naturally present, eg, at least two heavy chain portions, but two. Antibodies that do not contain complete heavy chains (eg, domain-deficient antibodies or minibodies), are multispecific modified to bind to different epitopes on two or more different antigens or a single antigen. It includes morphological antibodies (eg, bispecific, trispecific, etc.), heavy chain molecules linked to scFv molecules, and the like. In addition, the term "antibody variant" includes antibodies in a polyvalent form (eg, trivalent, tetravalent, etc.), antibodies that bind to three, four, or more copies of the same antigen.

As used herein, the term "valency" means the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on the target molecule. If the polypeptide contains more than one target binding site, each target binding site may specifically bind to the same or different molecule (eg, bind to a different ligand or different antigen, or to a different epitope on the same antigen). Can be). It is preferred that the binding polypeptide of interest has at least one binding site specific for the human antigen molecule.

The term "specificity" means the ability to specifically bind (eg, react) to a given target antigen (eg, a human target antigen). The binding polypeptide is unispecific and may contain one or more binding sites that specifically bind to the target, or the polypeptide is multispecific and specific to the same or different targets. It may contain two or more binding sites that bind specifically. In certain embodiments, the binding polypeptides of the invention are specific for two different (eg, non-overlapping) moieties of the same target. In certain embodiments, the binding polypeptides of the invention are specific for more than one target. Exemplary binding polypeptides (eg, antibodies) comprising antigen binding sites that bind to antigens expressed on tumor cells are known in the art and one or more from such antibodies. The CDR may be included in the antibody of the present invention.

The term "linking moiety" includes a moiety capable of linking an effector moiety to a binding polypeptide disclosed herein. The linking moiety may be selected so that the linking moiety can be cleaved (eg, enzymatically cleaved or pH sensitive) or cannot be cleaved. Exemplary concatenated portions are set forth in Table 2 herein.

As used herein, the term "effector moiety" refers to an agent having biological or other functional activity (eg, proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties, and fragments thereof). including. For example, a modified binding polypeptide comprising an effector moiety conjugated to a binding polypeptide has at least one additional function or property as compared to an unconjugated antibody. For example, conjugation of a cytotoxic agent (eg, an effector moiety) to a binding polypeptide has a second function (ie, in addition to antigen binding) to form a binding polypeptide having drug cytotoxicity. Bring. In another example, the conjugation of the second binding polypeptide to the binding polypeptide may confer additional binding properties. In certain embodiments, where the effector moiety is a genetically encoded therapeutic or diagnostic protein or nucleic acid, the effector moiety is a peptide synthesis or recombinant DNA process well known in the art. It can be synthesized or expressed by any of the above. In another embodiment, if the effector moiety is a non-genetically encoded peptide, or drug moiety, the effector moiety may be artificially synthesized or purified from a natural source. .. As used herein, the term "drug moiety" refers to anti-inflammatory agents, anti-cancer agents, anti-infective agents (eg, antifungal agents, antibacterial agents, antiparasitic agents, antiviral agents, etc.), and anesthetic therapeutic agents. including. In a further embodiment, the drug moiety is an anticancer or cytotoxic agent. Applicable drug moieties may also include prodrugs. Exemplary effector moieties are listed in Table 1 herein.

In certain embodiments, the "effector portion" includes a "targeted portion". As used herein, the term "targeted moiety" means an effector moiety that binds to a target molecule. Targeted moieties can include, without limitation, proteins, nucleic acids, lipids, carbohydrates (eg, glycans), and combinations thereof (eg, glycoproteins, glycopeptides, and glycolipids).

As used herein, the term "prodrug" is less active, less responsive, or less prone to side effects, can be enzymatically activated, or otherwise. Means the form of a precursor or derivative of a pharmaceutically active drug that can be converted in vivo to a more active form. Applicable prodrugs in the compositions of the present disclosure include, but are not limited to, phosphate-containing prodrugs, amino acid-containing prodrugs, and thiophosphate-containing prodrugs that can be converted into more active cytotoxic free drugs. Drugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and others. The 5-fluorouridine prodrug of. One of ordinary skill in the art can chemically modify the desired drug moiety or a prodrug thereof to make the reaction of this compound more convenient for the purpose of preparing the modified binding polypeptide of the present disclosure. Drug moieties also include derivatives of the drug moieties described herein, pharmaceutically acceptable salts, esters, amides, and ethers. Derivatives include modifications to the drugs identified herein that can or do not significantly reduce the desired therapeutic activity of the particular drug.

As used herein, the term "antineoplastic agent" includes agents that are detrimental to the growth and / or proliferation of neoplastic or tumor cells and may act to reduce, inhibit, or destroy malignant diseases. .. Examples of such agents include, but are not limited to, cell division inhibitors, alkylating agents, antibiotics, cytotoxic nucleosides, tubulin binding agents, hormones, hormone antagonists, cytotoxic agents and the like. Cytotoxicants include tomymycin derivatives, maytancin derivatives, cryptophycine derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptnigrine derivatives, auristatin derivatives, and duocarmycin derivatives. Any agent that acts to slow or slow the growth of immunoreactive cells or malignant cells is within the scope of the present disclosure.

As used herein, the term "antigen" or "target antigen" means a molecule or portion of a molecule that can be bound by the binding site of the binding polypeptide. The target antigen may have one or more epitopes.

II. Bundling Polypeptides In one aspect, the present disclosure provides binding polypeptides (eg, antibodies, antibody fragments, antibody variants, and fusion proteins) comprising glycosylated domains, eg, glycosylated constant domains. do. The binding polypeptides disclosed herein include any binding polypeptide, including domains with N-linked glycosylation sites. In certain embodiments, the binding polypeptide is an antibody, or fragment or derivative thereof. Any antibody from any source or species can be used in the binding polypeptides disclosed herein. Suitable antibodies include, without limitation, human antibodies, humanized antibodies, or chimeric antibodies.

In certain embodiments, the glycosylation domain is the Fc domain. In certain embodiments, the glycosylation domain is the natural glycosylation domain of N297.

In other embodiments, the glycosylation domain is a modified glycosylation domain. An exemplary modified glycosylation domain in the Fc domain comprises an asparagine residue at amino acid position 298 by EU numbering; and a serine or threonine residue at amino acid position 300 by EU numbering.

In the binding polypeptides disclosed herein, Fc domains from any immunoglobulin class (eg, IgM, IgG, IgD, IgA, and IgE) and species can be used. Chimeric Fc domains that include parts of Fc domains from different species or Ig classes can also be used. In certain embodiments, the Fc domain is the Fc domain of human IgG1. In the case of the Fc domain of human IgG1, the N-linked glycosylation consensus site (ie, X is other than proline) due to the mutation of the wild-type amino acid Kabat to asparagine at position 298 and Kabat to serine or threonine at position 300. It results in the formation of all amino acids, N-XT / S-sequon). However, for Fc domains of other species and / or Ig class or isotypes, those skilled in the art will be able to recreate the N-XT / S sequence if a proline residue is present, Kabat of the Fc domain. It will be understood that it may be necessary to mutate the 299th position of.

In other embodiments, the present disclosure provides binding polypeptides (eg, antibodies, antibody fragments, antibody variants, and fusion proteins) that include at least one CH1 domain with an N-linked glycosylation site. Such exemplary binding polypeptides can include, for example, a glycosylation site modified to position 114 by Kabat numbering.

CH1 domains from any immunoglobulin class (eg, IgM, IgG, IgD, IgA, and IgE) and species can be used in the binding polypeptides disclosed herein. Chimeric CH1 domains containing potions of CH1 domains from different species or classes of Ig can also be used. In certain embodiments, the CH1 domain is the CH1 domain of human IgG1. In the human IgG1 domain, mutation of the wild-type amino acid at position 114 to asparagine forms an N-linked glycosylation consensus site (ie, N-XT / S sequence, where X is any amino acid except proline). Is brought about. However, for other CH1 domains of other species and / or Ig class or isotypes, one of ordinary skill in the art would mutate positions 115 and / or 116 of the CH1 domain to give the N-XT / S sequence. It will be understood that it may be necessary to make.

In certain embodiments, the binding polypeptides of the present disclosure may comprise an antigen binding fragment of an antibody. The term "antigen-binding fragment" refers to an antibody that binds to an antigen or competes with an antibody that is intact with respect to antigen binding (ie, specific binding) (ie, with an intact antibody from which the antigen-binding fragment has been induced). Means a polypeptide fragment of an immunoglobulin or antibody. Antigen-binding fragments can be produced by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include Fv, Fab, Fab', and (Fab') 2. In a preferred embodiment, the antigen-binding fragment of the present disclosure is a modified antigen-binding fragment containing at least one modified glycosylation site. In one exemplary embodiment, the modified antigen-binding fragment of the present disclosure comprises the modified VH domain described above. In another exemplary embodiment, the modified antigen-binding fragment of the present disclosure comprises the modified CH1 domain described above.

In an exemplary embodiment, the binding polypeptide comprises a single chain variable region sequence (ScFv). The sequence of the single chain variable region comprises a single polypeptide having one or more antigen binding sites, such as the VL domain linked to the VH domain by a flexible linker. The ScFv molecule can be constructed with a VH-linker-VL configuration or a VL-linker-VH configuration. The flexible hinge connecting the VL domain and the VH domain constituting the antigen binding site preferably contains about 10 to about 50 amino acid residues. Connecting peptides are known in the art. The binding polypeptide of the invention may contain at least one scFv and / or at least one constant region. In one embodiment, the binding polypeptide of the present disclosure is a CH1 domain (eg, a CH1 domain containing an asparagine residue at position 114 of Kabat) and / or a CH2 domain (eg, an asparagine residue at position EU298, and EU300). It may contain at least one scFv linked or fused to an antibody or fragment containing (CH2 domain) containing a serine or threonine residue at the position.

In certain exemplary embodiments, the binding polypeptides of the present disclosure are made by fusing a DNA sequence encoding an antibody having a ScFv molecule (eg, a modified ScFv molecule), and are multivalent (eg, eg). It is a tetravalent) antibody. For example, in one embodiment, these sequences are such that the ScFv molecule (eg, a modified ScFv molecule) is linked to the Fc fragment of the antibody by a flexible linker (eg, ly / ser linker) at its N-terminus or C-terminus. It is combined like this. In another embodiment, the tetravalent antibody of the present disclosure is a ScFv-Fab in which a ScFv molecule is fused to a ligation peptide fused to a CH1 domain (eg, a CH1 domain containing an asparagine residue at position 114 of Kabat). It can be produced by constructing a tetravalent molecule.

In another embodiment, the binding polypeptide of the present disclosure is a modified minibody. The modified minibodies of the present disclosure each include a ScFv molecule (eg, a modified ScFv molecule comprising the modified VH domain described above) fused to the CH3 domain or a portion thereof by a connecting peptide 2 It is a dimer molecule that constitutes the polypeptide chain of a book. Minibodies can be made by constructing ScFv components and connecting peptide-CH3 components using the methods described in the art (eg, US Pat. No. 5,837,821). Or see WO94 / 09817Al). In another embodiment, a tetravalent minibody can be constructed. The tetravalent minibody can be constructed in the same manner as the minibody, except that the two ScFv molecules are linked using a flexible linker. The ligated scFv-scFv construct is then linked to the CH3 domain.

In another embodiment, the binding polypeptides of the present disclosure comprise bispecific antibodies. Bispecific antibodies each have a polypeptide similar to the scFv molecule, but are short (10) connecting both variable domains so that VL and VH domains on the same polypeptide chain cannot interact. A dimeric, tetravalent molecule that usually has a linker of amino acid residues (less than 1, preferably 1-5). Instead, the VL and VH domains of one polypeptide chain interact (respectively) with the VH and VL domains on the second polypeptide chain (see, eg, WO 02/02781). The bispecific antibodies of the present disclosure include scFv molecules fused to the CH3 domain.

In other embodiments, the binding polypeptide of the invention is a multispecific or multivalent antibody comprising one or more variable domains contiguously on the same polypeptide chain, eg, a tandem variable domain (eg, a tandem variable domain). TVD) Contains polypeptides. Exemplary TVD polypeptides include the "double head" or "dual Fv" configuration described in US Pat. No. 5,989,830. In the dual Fv configuration, the variable domains of two different antibodies are expressed in a tandem configuration on two separate chains (one heavy chain and one light chain), in this case one polypeptide chain. In addition, there are two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains continuously linked by a peptide linker (VL1-linker). -VL2). In the crossed double-head configuration, the variable domains of the two different antibodies are expressed in a tandem configuration on two separate polypeptide chains (one heavy chain and one light chain), in this case 1 In the book polypeptide chain, two VH domains are consecutively separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chains are continuously connected by a peptide linker in the reverse arrangement. Consists of complementary VL domains (VL2-linker-VL1). Additional antibody variants based on the "dual-Fv" format include dual-variable domain IgG (DVD-IgG) bispecific antibodies (see US Pat. No. 7,612,181) and TBT1 format (US2010). / 0226923A1). Functional bispecific without any further modification by adding constant domains to each chain of dual-Fv (CH1-Fc to heavy chain, kappa or lambda constant domain to light chain). Antibodies are delivered (ie, constant domains are clearly added to enhance stability).

In another exemplary embodiment, the binding polypeptide is a crossed double variable based on a "double head" configuration (see US201120251541A1, the full text of which is incorporated herein by reference). Includes bispecific antibodies with domain IgG (CODV-IgG). The CODV-IgG antibody variant is a single polypeptide chain with a VL domain (VL1-L1-VL2-L2-CL) that is continuously linked to the CL domain, and to the CH1 domain in consecutive opposite orientations. It has a second polypeptide chain with a connected complementary VH domain (VH2-L3-VH1-L4-CH1), where the polypeptide chains form crossed light chain-heavy chain pairs. In certain embodiments, the second polypeptide may be further linked to the Fc domain (VH2-L3-VH1-L4-CH1-Fc). In certain embodiments, the linker L3 is at least twice as long as the linker L1 and / or the linker L4 is at least twice as long as the linker L2. For example, L1 and L2 may be 1 to 3 amino acid residue lengths, L3 may be 2 to 6 amino acid residue lengths, and L4 may be 4 to 7 amino acid residue lengths. May be. Examples of suitable linkers are a single glycine (Gly) residue, a diglycine peptide (Gly-Gly), a tripeptide (Gly-Gly-Gly), and a peptide with four glycine residues (Gly-Gly-Gly-). Gly), a peptide with 5 glycine residues (Gly-Gly-Gly-Gly-Gly), a peptide with 6 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly), and a peptide with 7 glycine residues. (Gly-Gly-Gly-Gly-Gly-Gly-Gly), a peptide with 8 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly) is included. Other combinations of amino acid residues such as the peptide Gly-Gly-Gly-Gly-Ser and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser may be used.

In certain embodiments, the binding polypeptide comprises an immunoadhesin molecule (eg, the full text thereof) comprising a non-antibody binding region (eg, a receptor, a ligand, or a cell adhesion molecule) fused to an antibody constant region. See Ashkenazi et al., Methods, 1995, Vol. 8, (2), Nos. 104-115, which are incorporated herein by reference).

In certain embodiments, the binding polypeptide comprises an immunoglobulin-like domain. Suitable immunoglobulin-like domains are, but are not limited to, fibronectin domains (eg, Koide et al. (2007), Methods Mol. Biol., Vol. 352, 95-, the full text of which is incorporated herein by reference. See page 109, DARPin (see, eg, Stumpp et al. (2008), Drag Discov. Today, Volume 13 (15-16), pp. 695-701, the full text of which is incorporated herein by reference), Protein. Z domain of A (see Nygren et al. (2008), FEBS J., Vol. 275 (11), pp. 2668-76, the full text of which is incorporated herein by reference), lipocalin (eg, the full text thereof). Skerra et al. (2008), FEBS J., Vol. 275 (11), pp. 2677-83), Affiliins (eg, the full text of which is incorporated by reference. See Ebersbach et al. (2007), J. Mol. Biol., Vol. 372 (1), pp. 172-85), which are incorporated herein. See Krehenbrink et al. (2008), J. Mol. Biol., Vol. 383 (5), pp. 1058-68), Avimmers (eg, the full text of which is incorporated herein by reference. Incorporated, Silverman et al. (2005), Nat. Biotechnol., Vol. 23 (12), pp. 1556-61), Phynomers (eg, the full text of which is incorporated herein by reference. Grabulovski et al. (2007), J Biol Chem, Vol. 282 (5), pp. 3196-3204), and the Kunitz domain peptide (eg, the full text of which is incorporated herein by reference, Nixon. Et al. (2006), Curr Opin Drug Discov Devel, Vol. 9, (2), pp. 261-8).

III. N-linked glycans In certain embodiments, the binding polypeptide of the invention is N-linked via an asparagine residue to the glycosylation site in the polypeptide backbone of the binding polypeptide. Use type glycans. The glycosylation site may be natural or modified glycosylation site. Further or / or, the glycan may be a natural glycan or a modified glycan containing a non-natural linkage.

In certain exemplary embodiments, the binding polypeptide of the invention comprises the natural glycosylation site of the antibody Fc domain. This natural glycosylation site contains a wild-type asparagine residue at position 297 of the EU numbered Fc domain (N297). The natural N-linked glycans in this position are generally linked to the nitrogen group of the N297 side chain by β-glycosylamide linkage. However, other suitable linkages recognized in the art can also be used. In another exemplary embodiment, the binding polypeptide of the invention comprises one or more modified glycosylation sites. Such modified glycosylation sites are at asparagine residues that can be N-glycosylated by the cellular glycosylation enzyme of one or more wild amino acids in the polypeptide backbone of the binding polypeptide. Includes substitution. An exemplary modified glycosylation site of the invention comprises the introduction of an asparagine mutation at amino acid position 298 (298N) in the Fc domain or amino acid 114 (114N) in the CH1 domain.

Any type of naturally occurring or synthetic (ie, non-natural) N-linked glycans can be linked to the glycosylation site of the binding polypeptide of the invention. In certain embodiments, the glycan comprises a sugar (eg, a sugar residue located at the end of an oligosaccharide), the sugar being oxidized (eg, by periodic acid treatment or galactose oxidase) and an effector moiety (eg, by galactose oxidase). It is possible to generate a group suitable for conjugation to a reactive aldehyde group). Suitable oxidizable sugars include, but are not limited to, galactose and sialic acid (eg, N-acetylneuraminic acid). In certain embodiments, the glycan is a bifurcated glycan. In certain embodiments, glycans are naturally occurring mammalian sugar forms.

Glycosylation can be achieved by any means known in the art. In certain embodiments, glycosylation is achieved by expressing the binding polypeptide in cells capable of N-linked glycosylation. Any natural or modified cell (eg, prokaryote or eukaryote) can be used. Generally, mammalian cells are used to achieve glycosylation. N-glycans produced in mammalian cells are commonly referred to as complex, hypermanose, hybrid N-glycans (eg, Drickamer K, Taylor ME (2006), the full text of which is incorporated herein by reference). , Introduction to Glycobiology, 2nd Edition). These complex N-glycans have a structure with 2 to 6 external branches, typically having a sialyllactoseamine sequence linked to the inner core structure Man ₃ GlcNAc ₂ . The complex N-glycan ends with an oligosaccharide and has at least one, preferably at least two branches, alternating GlcNAc and galactose (Gal) residues, eg, NeuNAc-; NeuAcα2. , 6 GalNAcα1-; NeuAcα2,3 Galβ1,3 GalNAcα1-; and NeuAcα2,3 / 6 Galβ1,4 GlcNAcβ1 and the like. In addition, sulfate esters may be present on galactose, GalNAc, and GlcNAc residues. NeuAc may be O-acetylated or replaced with NeuGl (N-glycolylneuraminic acid). Complex N-glycans may have interchain substitutions of bisect-type GlcNAc and core fucose (Fuc).

Further, or / or, glycosylation can be achieved or modified in vitro by enzymatic means. For example, one or more glycosyltransferases may be used to add specific sugar residues to the natural or modified N-glycans of the binding polypeptide. May be used to remove unwanted sugars from N-linked glycans. Such enzymatic means are well known in the art (see, eg, WO 2007/005786, the full text of which is incorporated herein by reference).

IV. Immunological Effector Function and Fc Modification In certain embodiments, the binding polypeptide of the invention mediates one or more effector functions in an antibody constant region (eg, an IgG constant region, eg, a human IgG constant region). , For example, human IgG1 or IgG4 constant region). For example, the C1 complex can activate the complement system when bound to the antibody constant region. Activation of the complement system is important for opsonization and lysis of cellular pathogens. Activation of the complement system can also stimulate the inflammatory response and contribute to autoimmune hypersensitivity. In addition, the antibody binds to various cellular receptors by the Fc region (the Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell). There are a number of Fc receptors specific for different classes of antibodies, such as IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). When an antibody binds to an Fc receptor on the cell surface, it phagocytoses and destroys particles coated by the antibody, clearance of the immune complex, and lysis of target cells coated by the antibody by killer cells (antibody-dependent cell-mediated cytotoxicity or ie. A number of important and diverse biological responses are elicited, including (called ADCC), release of inflammatory mediators, placement of the placenta, and regulation of immunoglobulin production. In a preferred embodiment, the binding polypeptide of the invention (eg, an antibody or antigen-binding fragment thereof) binds to the Fc-gamma receptor. In an alternative embodiment, the binding polypeptide of the invention may comprise a constant region lacking one or more effector functions (eg ADCC activity) and / or may not bind to the Fcγ receptor.

Certain embodiments of the invention include reduced or enhanced effector function, the ability to dimerize non-covalently, and the site of tumors when compared to all unchanged antibodies of approximately the same immunogenicity. At least one amino acid in one or more constant region domains is deleted to provide the desired biochemical features, such as increased ability to localize, reduced serum half-life, or increased serum half-life. , Or otherwise modified antibody. For example, certain antibodies for use in the diagnostic and therapeutic methods described herein contain a polypeptide chain similar to an immunoglobulin heavy chain, but at least a portion of one or more heavy chain domains. Is a domain-deficient antibody in which is deleted. For example, in certain antibodies, one domain of the constant region of the modified antibody is deleted, eg, all or part of the CH2 domain.

In another predetermined embodiment, the binding polypeptide comprises a constant region derived from various antibody isotypes (eg, a constant region from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the binding polypeptide is a chimeric hinge (ie, a hinge comprising a hinge moiety derived from the hinge domain of various antibody isotypes, eg, an upper hinge domain from an IgG4 molecule and an IgG1 central hinge domain). including. In one embodiment, the binding polypeptide comprises the Fc region from a human IgG4 molecule or a portion thereof, and the Ser228Pro mutation (EU numbering) in the core hinge region of the molecule.

In certain embodiments, the Fc moiety may be mutated to increase or decrease effector function using techniques known in the art. For example, deletion or inactivation of the constant region domain (by point mutation or other means) can reduce Fc receptor binding of the circulating modified antibody, thereby increasing tumor localization. In other cases, modification of the constant region consistent with the present invention relaxes complement binding, thus reducing serum half-life and non-specific association of conjugated cytotoxicities. Still other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties, these modifications enhancing localization by increasing the specificity or flexibility of the antigen. The resulting physiological profile, bioavailability, and other biochemical effects of modification, such as tumor localization, biodistribution, and serum half-life, are well known without undue experimentation. It can be easily measured and quantified using immunological techniques.

In certain embodiments, the Fc domain used in the antibodies of the invention is an Fc variant. As used herein, the term "Fc variant" means an Fc domain having at least one amino acid substitution as compared to the wild-type Fc domain from which said Fc domain is derived. For example, if the Fc domain is derived from a human IgG1 antibody, the Fc variant of the Fc domain of human IgG1 contains at least one amino acid substitution compared to the Fc domain.

Amino acid substitutions (s) of Fc variants may be located at any position within the Fc domain (ie, amino acid positions of any EU regulation). In one embodiment, the Fc variant comprises a substitution of an amino acid position located at or in part of the hinge domain. In another embodiment, the Fc variant comprises a substitution of an amino acid position located in or part of the CH2 domain. In another embodiment, the Fc variant comprises the substitution of an amino acid position located in or part of the CH3 domain. In another embodiment, the Fc variant comprises the substitution of an amino acid position located in or part of the CH4 domain.

The binding polypeptides of the invention can be Fc variants recognized in all arts known to result in an improvement (eg, reduction or enhancement) in effector function and / or FcR binding. The Fc variants are, for example, the International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 /, each of which is incorporated herein by reference in its entirety. 09560A2, WO00 / 32767A1 WO05 / 070963A1, WO05 / 077981A2, WO05 / 0992925A2, WO05 / 123780A2, WO06 / 09147A1, WO06 / 047350A2 and WO06 / 085967A2, or US Pat. Nos. 5,648,260, 5,739,277, 5 , 834,250, 5,869,046, 6,096,871, 6,121,022, 6,194,551, 6,242,195, 6,277 , 375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083, It may contain any one of the amino acid substitutions disclosed in 784. In one exemplary embodiment, the binding polypeptide of the invention may comprise an Fc variant (eg, H268D or H268E) comprising an amino acid substitution at position EU268. In another exemplary embodiment, the binding polypeptide of the invention may comprise an amino acid substitution at position EU239 (eg, S239D or S239E) and / or position EU332 (eg, I332D or I332Q). ..

In certain embodiments, the binding polypeptide of the invention may comprise an Fc variant comprising an amino acid substitution that alters the antigen-independent effector function of the antibody, particularly the circulating half-life of the binding polypeptide. Such binding polypeptides show either increased or reduced binding to FcRn when compared to these non-substitution binding polypeptides, thus providing increased or reduced serum half-life, respectively. Have. The serum half-life of Fc variants with improved affinity for FcRn is predicted to be longer, and such molecules should have a longer half-life of the antibody to be administered, such as in the treatment of chronic diseases or disorders. It is advantageously applied in the method of treating mammals. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also circulating, for example, in vivo diagnostic imaging, or the first antibody. It is useful for administration to mammals when shortening the circulation time may be advantageous, such as in situations where there are toxic side effects when present in a long period of time. Fc variants with reduced FcRn binding affinity are also less likely to translocate the placenta and are therefore also useful in treating diseases or disorders in pregnant women. In addition, other applications where reduced FcRn binding affinity may be desirable include applications localized to the brain, kidney, and / or liver. In one exemplary embodiment, the modified binding polypeptide of the invention (eg, an antibody or antigen-binding fragment thereof) exhibits reduced transport across the epithelium of the glomerulus from the vasculature. In another embodiment, the modified binding polypeptide of the invention (eg, an antibody and its antigen-binding fragment) is transported from the brain across the blood-brain barrier (BBB) into the interstitial space of blood vessels. Shows a reduction. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of the Fc domain. The FcRn binding loop consists of amino acid residues 280-299 (by EU numbering). Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication No. WO 05/047327, which is incorporated herein by reference in its entirety. In an exemplary predetermined embodiment, the binding polypeptide of the invention (eg, an antibody or antigen-binding fragment thereof) is one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D ( Contains Fc domains with EU numbering). In yet another exemplary embodiment, the binding molecule of the invention is a double mutant H4.
Includes a human Fc domain with 33K / N434F (see, eg, US Pat. No. 8,163,881).

In other embodiments, the binding polypeptide for use in the diagnostic and therapeutic methods described herein is a constant region, eg IgG1 or IgG4 heavy chain determination, modified to reduce or eliminate glycosylation. Has a normal region. For example, the binding polypeptide of the invention (eg, an antibody or antigen-binding fragment thereof) may also include an Fc variant comprising an amino acid substitution that alters glycosylation of the antibody Fc. For example, glycosylation of the Fc variant may be reduced (eg, N or O-linked glycosylation). In an exemplary embodiment, the Fc variant comprises reducing glycosylation of N-linked glycans commonly found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within the glycosylation motif, such as an N-linked glycosylation motif comprising the amino acid sequence NXT or NXS. In one particular embodiment, the antibody comprises an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody comprises an IgG1 or IgG4 constant region containing S228P and T299A mutations (EU numbering).

Exemplary amino acid substitutions that impart reduction or modification of glycosylation are disclosed in International PCT Publication No. WO 05/018572, the full text of which is incorporated herein by reference. In a preferred embodiment, the binding polypeptide of the invention is modified to eliminate glycosylation. Such binding polypeptides can be referred to as "agly" binding polypeptides (eg, "agri" antibodies). Without being bound by theory, it is believed that "agri" -binding polypeptides may have an in vivo improved safety and stability profile. The agri-binding polypeptide may be of any isotype or subclass thereof, such as IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the agri-binding polypeptide comprises a non-glycosylated Fc region of an IgG4 antibody that lacks Fc-effector function and is therefore of Fc-mediated toxicity to normal critical organs expressing IL-6. Possibility is ruled out. In yet another embodiment, the binding polypeptide of the invention comprises a modified glycan. For example, the antibody may have a small number of fucose residues on the N-glycan of Asn297 in the Fc region, i.e., afucosylated. Non-fucosylation increases FcγRII binding on NK cells and potentially increases ADCC. Diabodies containing anti-IL-6 scFv and anti-CD3 scFv have been shown to induce the killing of IL-6 expressing cells by ADCC. Thus, in one embodiment, non-fucosylated anti-IL-6 antibodies are used to target and kill IL-6 expressing cells. In another embodiment, the binding polypeptide may have a modified number of sialic acid residues on the N-glycan of Asn297 in the Fc region. Numerous techniques recognized in the art are available for making "agri" antibodies or antibodies with altered glycans. For example, a genetically modified host cell (eg, modified yeast, eg, Pichia or CHO cell) having a modified glycosylation pathway (eg, glycosyltransferase deletion) is used to generate such antibodies. can do.

V. Effector Subsections In certain embodiments, the binding polypeptides of the present disclosure include effector moieties (eg, drug moieties and targeting moieties). In general, these effector moieties are conjugated (either directly or via a linker moiety) to N-linked glycans on the binding polypeptide (eg, N-linked glycans are N298 in the CH2 domain). (EU numbering) and / or linked to N114 (Kabat numbering) of the CH1 domain). In certain embodiments, the binding polypeptide is a full-length antibody comprising two CH1 domains with glycans at position Kabat 114, where both glycans are conjugated to one or more effector moieties. There is.

Any effector moiety can be added to the binding polypeptide disclosed herein. The effector moiety preferably adds non-natural function to the modified antibody or fragment thereof without significantly altering the intrinsic activity of the binding polypeptide. The effector portion may be, for example, but not limited to, a therapeutic agent or a diagnostic agent. The modified binding polypeptides (eg, antibodies) of the present disclosure may contain one or more effector moieties, which may be the same or different.

In one embodiment, the effector portion is of formula (I) :.
H ₂ NQ-CON-X
Equation (I)
May be
During the ceremony
A) Q is NH or O,
B) CON is a connection part,
C) X is an effector moiety (eg, a therapeutic or diagnostic agent as defined herein).

The connecting portion connects the therapeutic agent to _H2NQ- . The connecting portion is, for example, an alkylenyl component, a polyethylene glycol component, a poly (glycine) component, a poly (oxazoline) component, a carbonyl component, a cysteine amide-derived component, or a valine-derived component that couples to citrulline. , And any suitable component known to those of skill in the art, such as components derived from 4-aminobenzyl carbamate, or any combination thereof.

In another embodiment, the effector portion of formula (I) is the formula (Ia) :.
H ₂ NQ-CH ₂ -C (O) -Z-X
Equation (Ia)
May be
During the ceremony
A) Q is NH or O,
B) Z is -Cys- (MC) _a- (VC) _b- (PABC) _c- (C ₁₆ H ₃₂ O ₈ C ₂ H ₄ ) _f .
During the ceremony
i. Cys is a constituent derived from cysteine amide.
ii. MC is a component derived from maleimide and is a component.
iii. VC is a valine-derived component that couples to citrulline.
iv. PABC is a component derived from 4-aminobenzyl carbamate and is a component.
v. X is an effector portion (eg, a therapeutic or diagnostic agent as defined herein).
vi. a is 0 or 1 and is
vii. b is 0 or 1 and is
viii. c is 0 or 1 and
ix. f is 0 or 1.

を有するエフェクター部分の１つまたはそれ以上の部分を指す場合がある。 The "component derived from cysteine amide" is an adhesion point to H2N-Q- _{CH2 -} C (O)-. In one embodiment, the "components derived from cysteine amide" is the structure:

May refer to one or more of the effector portions with.

In one embodiment, the "Cys" component of the effector portion may include one such portion. For example, the following structure shows an effector portion having one such portion (in the formula, the "Cys" component is indicated by a dotted box):

In another embodiment, the "Cys" component of the effector moiety may include more than one such moiety. For example, the following part includes two such parts.

As can be seen from the structure, the "Cys" component carries- (MC) _a- (VC) _b- (PABC) _c- (C ₁₆ H ₃₂ O ₈ C ₂ H ₄ ) _f -X groups.

を有するエフェクター部分のあらゆる部分を指す場合があり、式中、ｄは２から５の整数である。エフェクター部分におけるあらゆるＣｙｓ－（ＭＣ）_ａ－（ＶＣ）_ｂ－（ＰＡＢＣ）_ｃ－（Ｃ_１６Ｈ_３２Ｏ_８Ｃ_２Ｈ_４）_ｆ－Ｘ基に含まれるＭＣ構成成分の数は、下付き
数字「ａ」によって示され、０または１であり得る。一実施形態において、ａは１である。別の一実施形態において、ｂは０である。 In one embodiment, the expression "maleimide-derived constituents" refers to structure:

May refer to any part of the effector portion with, where d is an integer from 2 to 5. The number of MC components contained in all Cys- (MC) _a- (VC) _b- (PABC) _c- (C ₁₆ H ₃₂ O ₈ C ₂ H ₄ ) _f -X groups in the effector section is a subscript number. It is indicated by "a" and can be 0 or 1. In one embodiment, a is 1. In another embodiment, b is zero.

In one embodiment, the "Cys" component may be connected to the "MC" component via a sulfur atom in the "Cys" component, as shown by the dotted box in the structure below:

In one embodiment, the expression "valine-derived component that couples with citrulline" may refer to any part of an effector portion having the following structure:

The number of VC components contained in any Cys- (MC) _a- (VC) _b- (PABC) _c- (C ₁₆ H ₃₂ O ₈ C ₂ H ₄ ) _f -X group in the effector portion is a subscript. It is indicated by "b" and may be 0 or 1. In one embodiment, b is 1. In another embodiment, b is zero.

In one embodiment, the expression "components derived from 4-aminobenzyl carbamate" may refer to any portion of an effector moiety having the following structure:

The number of PABC components contained in any Cys- (MC) _a- (VC) _b- (PABC) _c- (C ₁₆ H ₃₂ O ₈ C ₂ H ₄ ) _f -X group in the effector portion is a subscript. It is indicated by "c" and may be 0 or 1. In one embodiment, c is 1. In another embodiment, c is 0.

In one embodiment, "C ₁₆ H ₃₂ O ₈ C ₂ H ₄ " refers to the following structure:

Number of units of C ₁₆ H ₃₂ O ₈ contained in any Cys- (MC) _a- (VC) _b- (PABC) _c- (C ₁₆ H ₃₂ O ₈ C ₂ H ₄ ) _f -X group in the effector portion Is indicated by the subscript "f". In one embodiment, f is 1. In another embodiment, f is zero.

In one embodiment, a is 1, b is 1, c is 1, and f is 0.

a) Therapeutic effector moieties In certain embodiments, the binding polypeptides of the present disclosure are conjugated to a therapeutic agent, eg, a drug moiety (or a prodrug thereof), or an effector moiety comprising a radiolabeled compound. .. In one embodiment, the therapeutic agent is cytotoxic. Exemplary cytotoxic therapeutic agents are listed in Table 1 herein.

Further exemplary drug moieties include anti-inflammatory agents, anti-cancer agents, anti-infective agents (eg, antifungal agents, antibacterial agents, antiparasitic agents, antiviral agents, etc.), and anesthetic therapeutic agents. In a further embodiment, the drug moiety is an anti-cancer agent. Exemplary anti-cancer agents are, but are not limited to, cell division inhibitors, enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin-binding or tubulin inhibitors, proteasome inhibitors, hormones and hormones. Includes antagonists, angiogenesis inhibitors, etc. Exemplary cell division-inhibiting anticancer agents include alkylating agents, eg, drugs of the anthracycline family (eg, adriamycin, carminomycin, cyclosporine A, chloroquine, metopterin, mitramycin, porphyromycin, streptnigrin, porphyro). Includes mycin, anthracenedione, and aziridine). Other anticancer agents that inhibit cell division include DNA synthesis inhibitors (eg methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cisplatin β-D-arabino). Furanoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, gancyclovir, hydroxyurea, actinomycin D, and mitomycin C), DNA intercalators or cross-linking agents (eg, bleomycin, carboplatin, carmustine, chlorambusyl, cyclophosphamide). Famide, cis-diamine platinum (II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcriptional regulators (eg, actinomycin D, daunorbisin, doxorubicin, homoharingtonin, and Idalbisin) is included. Other exemplary cell division inhibitors applicable in the present disclosure are ansamicin benzoquinone, quinonoid derivatives (eg, quinolone, genistein, bactaxiclin), busulfan, ifosfamide, chlormethine, triaziquone, diaziquone (eg, quinolone, ifosfamide, chlormethine). diaziquone), carbaziruquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (eg, carmustine, lomustine, semustine).

Exemplary cytotoxic nucleoside anti-cancer agents include, but are not limited to, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine. Exemplary anticancer tuberin binding agents are, but are not limited to, taxoids (eg, paclitaxel, docetaxel, taxane), nocodazole, lysoxins, drastatins (eg, drastatin-10, -11, or -15), Exemplary include corhitin and corhitinoids (eg ZD6126), combretastatins (eg combretastatin A-4, AVE-6032, and vinca alkaloids (eg vinblastine, vincristine, bindesin, and vinorelbine)). Anticancer hormones and hormonal antagonists are, but are not limited to, corticosteroids (eg, prednison), progestin (eg, hydroxyprogesterone or medroprogesterone), estrogen (eg, diethylstilvestrol), anti-estrogen (eg, eg, diethylstilvestrol). Tamoxyphene), androgen (eg, testosterone), aromatase inhibitor (eg, aminoglutetimide), 17- (allylamino) -17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, breferdin Exemplary anticancer anti-angiogenic compounds include A, simethidine, dichloromethylene-diphosphonic acid, leuprolide (leuprolerin), luteinizing hormone-releasing hormone, piffithrine a, rapamycin, sex-hormone-binding globulin, and tapsigardin. Includes, but is not limited to, angiostatins K1-3, DL-a-difluoromethyl-ornithine, endostatin, fumagirin, genistein, minocycline, staulosporin, and (±) -salidomide.

Exemplary anti-cancer enzyme inhibitors are, but are not limited to, S (±) -camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenzimidazole 1-β-D ribofuranoside, etoposide, forme. Includes Stan, Phostriecin, Hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatin), mevinolin, tricostatin A, tyrohostin AG34, and tyrohostin AG879.

Exemplary anticancer gene regulators are, but are not limited to, 5-aza-2'-deoxycitidine, 5-azacitidine, choleciferol (vitamin D3), 4-hydroxytamoxyphene, melatonin, mifepri. Includes stone, laroxifene, trans-retinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxyphene, and troglycazone.

Other preferred classes of anti-cancer agents include, but are not limited to, for example, the pteridine family of drugs, diyne, and podophyllotoxin. Particularly useful types of these classes include, for example, metopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosideine, vindesine, leurocine and the like.

Yet other anti-cancer agents applicable in the teachings herein are auristatin (eg, auristatin E and monomethyl auristatin E), geldanamycin, calikeamycin, glamicidin D, maytancinoids (eg, maytancin), and the like. Neocartinostatin, topotecan, taxan, cytocaracine B, ethidium bromide, emetin, teniposide, corhitin, dihydroxyanthrasindione, mitoxantrone, prokine, tetracaine, lidocaine, propranolol, puromycin, and their analogs or homologues. including.

Still other anti-cancer agents applicable as taught herein are tomymycin derivatives, maytancin derivatives, cryptophycin derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptnigrine derivatives, auristatin derivatives, and duocarmycin derivatives. including.

Another class of applicable anti-cancer agents that can be used as a drug moiety is a radiosensitizing drug that can effectively target tumors or immunoreactive cells. Such drug moieties increase the efficacy of radiation therapy by increasing sensitivity to ionizing radiation. Although not limited to theory, antibodies modified with a radiosensitizing drug moiety and internalized in tumor cells deliver the radiosensitizer closer to the nucleus, where radiosensitization is maximal. Is expected to be. Antibodies that have lost the radiosensitizer portion are rapidly removed from the blood, the remaining radiosensitizer is localized to the target tumor, and uptake into normal tissue is minimized. After being removed from the blood, adjunctive radiation therapy can be given by topical radiation to the tumor, direct implantation of the tumor, or radioimmunotherapy with the same modified antibody.

In one embodiment, the therapeutic agent comprises a radionuclide or a radiolabel with high energy ionizing radiation, which can cause multiple strand breaks in the DNA of the nucleus, resulting in cell death. Exemplary high energy radionuclides include 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, and 188Re. These isotopes typically produce high-energy alpha or beta particles with short orbital lengths. Such radionuclides kill cells that are highly proximal, such as conjugate-attached or invaded neoplastic cells. They have little or no effect on delocalized cells and are essentially non-immunogenic. Alternatively, high energy isotopes can be produced by heat radiation of otherwise stable isotopes, as in the case of boron neutron capture therapy (Guan et al., PNAS, Vol. 95, pp. 13206-10, 1998). ).

In one embodiment, the therapeutic agent is selected from MMAE, MMAF, and PEG8-Do110.

Exemplary therapeutic effector moieties include the following structures:

In one embodiment, the effector portion is selected from:

In certain embodiments, the effector moiety comprises more than one therapeutic agent. These plurality of therapeutic agents may be the same or different.

b) Diagnostic effector moiety In certain embodiments, the binding polypeptide of the present disclosure is conjugated to an effector moiety containing a diagnostic agent. In one embodiment, the diagnostic agent is a detectable small molecule label such as biotin, fluorophore, chromophore, spin resonance probe, or radiolabel. Exemplary fluorophores include fluorescent dyes (eg, fluorescein, rhodamine, etc.), and other fermentation molecules (eg, luminal). Fluorophores are environmentally sensitive so that their fluorescence changes when located close to one or more residues in a modified binding polypeptide that undergo structural changes upon binding to a substrate. May be (eg, a dansyl probe). Exemplary radiolabels are small molecules containing atoms with one or more hyposensitive nuclei (such as 13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe, 64Cu, 68Ga, 111In). including. The radionuclide is a gamma, photon, or positron-emitting radionuclide with a suitable half-life to allow activity or detection after the elapsed time between administration and localization to the imaging site. Is preferable.

In one embodiment, the diagnostic agent is a polypeptide. An exemplary diagnostic polypeptide is
Includes enzymes with fluorescence-generating or dye-producing activity, such as the ability to cleave a fluorophore or chromophore-forming substrate as a product (ie, a reporter protein such as luciferase). Other diagnostic proteins may have inherent fluorescence-generating or pigment-producing activity (eg, bioluminescent bioluminescent aequorin proteins from bioluminescent marine organisms), or 1 It may contain proteins containing one or more low energy radioactive nuclei (13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe, 64Cu, 68Ga, 111In, etc.).

With respect to the use of radiolabeled conjugates in combination with the present disclosure, the binding polypeptides of the present disclosure may be labeled directly (eg, by iodination) or indirectly by the use of chelating agents. good. Both the terms "indirect labeling" and "indirect labeling approach" used herein include a chelating agent covalently attached to a binding polypeptide and at least one radionuclide. It means that it is associated with a chelating agent. Such chelating agents are typically referred to as bifunctional chelating agents because they bind to both polypeptides and radioisotopes. Exemplary chelating agents include 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (“MX-DTPA”) and cyclohexyldiethylenetriaminepentaacetic acid (“CHX-DTPA”) derivatives. Other chelating agents include P-DOTA and EDTA derivatives. Radionuclides particularly preferred for indirect labeling include 111In and 90Y. Most imaging tests utilize 5 mCi 111In-labeled antibody, because this dose is safe, imaging efficiency is increased compared to low doses, and optimal imaging is 3 to 6 days after antibody administration. Because it will occur later. For example, Murray, (1985), J. Mol. Nuc. Med. , Vol. 26, p. 3328, and Carragullo et al. (1985), J. Mol. Nuc. Med. , Vol. 26, p. 67. A particularly preferred radionuclide for direct labeling is 131I. Those skilled in the art will appreciate that non-radioactive conjugates can be assembled according to the agent selected for the conjugate.

In certain embodiments, the diagnostic effector portion is a FRET (Fluorescence Resonance Energy Transfer) probe. FRET is used in various diagnostic applications such as cancer diagnosis. The FRET probe may include a cleavable linker (enzyme-sensitive or pH linker) that connects the donor and acceptor moieties of the FRET probe, and cleavage results in enhanced fluorescence (including near-infrared light) (including near-infrared light). For example, A. Cobos-Correa et al., Membrane-bound FRET probe visions MMP12 activity in plummonary inframmation, Nature Chemical Biology (2009), pp. 63 Elastase Activity with Radiometic Fluorescent Reports (2012) Angew. Chem. Int. Ed., Vol. 51, pp. 6258-6261).

In one embodiment, the effector portion is selected from:

c) Functionalized effector moiety In a given embodiment, the effector moiety of the present invention may be functionalized so as to include additional groups in addition to the effector moiety itself. For example, the effector moiety may include a cleavable linker that releases the effector moiety from the binding polypeptide under certain conditions. In an exemplary embodiment, the effector moiety may contain a linker that is cleaved by cellular enzymes and / or is pH sensitive. Further, or / or, the effector moiety may contain a disulfide bond that is cleaved by intracellular glutathione when taken up into the cell. Exemplary disulfide and pH sensitive linkers are shown below:

In yet other embodiments, the effector moieties may include hydrophilic and biocompatible moieties such as poly (glycine), poly (oxazoline), or PEG moieties. An exemplary structure (“Y”) is provided below:

In certain embodiments, the effector moiety comprises an aminooxy group that promotes conjugation to the binding polypeptide by stable oxime binding. Exemplary effector moieties, including aminooxy groups, are listed in Table 2 herein.

In other embodiments, the effector moiety comprises a hydrazide and / or N-alkylated hydrazine group to facilitate conjugation to the binding polypeptide by stable hydrazone binding. Exemplary effector moieties containing aminooxy groups are listed in Table 14 herein.

d) Targeted moiety In certain embodiments, the effector moiety comprises a targeted moiety that specifically binds to one or more target molecules. Without limitation, any type of targeted moiety can be used, including proteins, nucleic acids, lipids, carbohydrates (eg, glycans), and combinations thereof (eg, glycoproteins, glycopeptides, and glycolipids). In certain embodiments, the targeting moiety is a carbohydrate or glycopeptide. In certain embodiments, the targeting moiety is a glycan. The targeting moiety may be a naturally occurring or non-naturally occurring molecule.

VI. Conjugation of Effector Substrate to Binding Polypeptide In certain embodiments, the effector moiety is a modified binding polypeptide (eg, N114 of the CH1 domain of an antibody modified glycan, or N297 of an antibody F domain. Is conjugated (either directly or via a linker moiety) to an oxidized glycan (eg, an oxidized N-linked glycan) (which is a natural glycan). The term "oxidized glycan" means that the alcohol substituent on the glycan has been oxidized to a carbonyl substituent. The carbonyl substituent can react with a suitable nitrogen nucleophile to form a carbon-nitrogen double bond. For example, when a carbonyl group reacts with an aminooxy group or a hydrazine group, an oxime or hydrazine is formed, respectively. In one embodiment, the carbonyl substituent is an aldehyde. Suitable oxidized glycans include oxidized galactose and oxidized sialic acid.

In one embodiment, the modified polypeptide of formula (II) is of formula (II) :.
Ab (Gal-C (O) H) _x (Gal-Sia-C (O) H) _y
Equation (II)
Can be shown in
During the ceremony
A) Ab is an antibody, or other binding polypeptide as defined herein.
B) Gal is a component derived from galactose and is a component.
C) Sia is a component derived from sialic acid and is a component.
D) x is 0 to 5,
E) y is 0 to 5,
At least one of x and y is not zero.

Any chemical reaction recognized in the art can be used to conjugate effector moieties (eg, effector moieties including linker moieties) to glycans (eg, Hermanson, the entire text of which is incorporated herein. , GT, Bioconjugate Techniques., Academic Press (1996)). In certain embodiments, the sugar residues of glycans (eg, sialic acid or galactose residues) are first oxidized (eg, using sodium periodate treatment of sialic acid or galactose oxidase treatment of galactose). Produces aldehyde groups. This aldehyde group is reacted with the effector moiety of an aminooxy group or a hydrazine group to form an oxime or a hydrazone linker, respectively. Exemplary methods using this general reaction scheme are described in Examples 10-15.

In certain embodiments, the native or modified glycans of the binding polypeptide are first pretreated in vitro with a glycosyltransferase to provide a terminal sugar residue with appropriate reactivity. For example, sialylation may be initially achieved with a combination of galactosyltransferase (GalT) and sialyltransferase (SialT). In certain embodiments, bifurcated glycans lacking galactose (G0F or G0) or containing only one galactose (G1F or G1) are conjugated to a higher order galactosylated or sialylated structure (G1F). , G1, G2F, G2, G1S1F, G1S1, G2S1F, G2S1, G2S2F, or G2S2).

An exemplary conjugate scheme for producing sialylated complex carbohydrates is shown in FIG. 25C. Sialic acid residues are enzymatically and site-specificly introduced into antibody glycans (eg, Asn-297 native glycans) using a combination of galactosyltransferase (GalT) and sialyltransferase (SialT). A drug-linker (eg, an aminooxy drug) for continuing to oxidize the introduced sialic acid residue with a low concentration of sodium periodate to produce an antibody drug conjugate (ADC) (eg, an oxime-bound ADC). A reactive sialic acid having appropriate reactivity with the linker) is obtained. By controlling the number of glycans and the number of sial residues by in vitro reconstruction, one of ordinary skill in the art can have precise control over the drug-antibody ratio (DAR) of the ADC. For example, when approximately one sialic acid is added on a single bifurcated glycan (A1F) in each heavy chain, an antibody or binding polypeptide having a DAT of 2 can be uniformly obtained.

VII. Modified Binding Polypeptides In certain embodiments, the present invention is an oxidation of a modified binding polypeptide (eg, a modified glycan of N114 in the antibody CH1 domain or a natural glycan of N297 in the antibody F domain). Provided are modified polypeptides which are products obtained by conjugating an effector moiety (either directly or by a linker moiety) to a glycan (eg, an oxidized N-linked glycan).

In one embodiment, the binding polypeptide is of formula (III) :.
Ab (Gal-C (H) = NQ-CON-X) _x (Gal-Sia-C (H) = NQ
-CON-X) _y
Equation (III)
Can be shown in
During the ceremony
A) Ab is an antibody defined herein and is an antibody.
B) Q is NH or O,
C) CON is the connection portion specified herein.
D) X is a therapeutic or diagnostic agent as defined herein.
E) Gal is a component derived from galactose and is a component.
F) Sia is a component derived from sialic acid and is a component.
G) x is 0 to 5,
H) y is 0 to 5,
At least one of x and y is not zero.

In one embodiment, the binding polypeptide is of formula (III) :.
Ab (Gal-C (H) = N-Q-CH ₂ -C (O) -Z-X) _x (Gal-Sia-C (H) = N-Q-CH ₂ -C (O) -Z- X) _y
Equation (IIIa)
Can be shown in
During the ceremony
A) Ab is an antibody and
B) Q is NH or O,
C) Z is Cys- (MC) _a- (VC) _b- ( _PABC ) _c- (C ₁₆ H ₃₂ O ₈ C ₂ H ₄ ) f-.
During the ceremony
i. Cys is a constituent derived from cysteine amide.
ii. MC is a component derived from maleimide and is a component.
iii. VC is a component derived from valine coupled with citrulline.
iv. PABC is a component derived from 4-aminobenzyl carbamate and is a component.
v. X is an effector portion (eg, a therapeutic or diagnostic agent as defined herein).
vi. a is 0 or 1 and is
vii. b is 0 or 1 and is
viii. c is 0 or 1 and
ix. f is 0 or 1 and is
D) X is a therapeutic agent as defined herein.
E) Gal is a component derived from galactose and is a component.
F) Sia is a component derived from sialic acid and is a component.
G) x is 0 to 5,
H) y is 0 to 5,
At least one of x and y is not zero.

It should be understood that formula (III) does not attempt to indicate that the antibody, Gal substituent, and Gal-Sia substituent are chain-linked. Instead, if such substituents are present, the antibody is directly linked to each substituent. For example, the binding polypeptide of formula (III) of formula (III) where x is 1 and y is 2 may have the configuration shown below:

The CON substituents in formula (III) and the constituents thereof are as described with respect to formula (I) for the effector moiety.

In one embodiment, Q is NH. In another embodiment, Q is O.

In one embodiment, x is 0.

The antibody Ab of formula (III) may be any suitable antibody described herein.

In one embodiment, a method for preparing a binding polypeptide of formula (III) is provided, the method of which is formula (I) :.
NH ₂ -Q-CON-X
Equation (I)
[During the ceremony,
A) Q is NH or O,
B) CON is a connection part,
C) X is an effector moiety (eg, a therapeutic or diagnostic agent as defined herein)]
The effector part of the formula (II):
Ab (OXG) _r
Equation (II)
[During the ceremony,
A) OXG is an oxidized glycan,
B) r is selected from 0 to 4]
Includes reacting with the modified antibody of.

In one embodiment, a method for preparing a binding polypeptide of formula (III) is provided, wherein the method is of formula (I) :.
NH ₂ -Q-CON-X
Equation (I)
[During the ceremony,
A) Q is NH or O,
B) CON is a connection part,
C) X is an effector moiety (eg, a therapeutic or diagnostic agent as defined herein)]
The effector part of the formula (IIa):
Ab (Gal-C (O) H) _x (Gal-Sia-C (O) H) _y
Equation (IIa),
[During the ceremony,
A) Ab is an antibody described herein.
B) Gal is a component derived from galactose and is a component.
C) Sia is a component derived from sialic acid and is a component.
D) x is 0 to 5,
E) y is 0 to 5,
At least one of x and y is not 0]
Includes reacting with the modified antibody of.

VII. Methods of Treating with Modified Antibodies In one aspect, the invention provides a method of treating or diagnosing a patient in need thereof, comprising administering an effective amount of the binding polypeptide disclosed herein. .. Preferred embodiments of the present disclosure provide kits and methods for diagnosing and / or treating disorders such as neoplastic disorders in mammalian subjects in need of such treatment. The subject is preferably a human.

The binding polypeptides of the present disclosure are useful in a number of different applications. For example, in one embodiment, the binding polypeptide of interest is useful for reducing or eliminating cells carrying an epitope that is recognized by binding to the binding domain of the binding polypeptide. In another embodiment, the binding polypeptide of interest is effective in reducing or removing the concentration of soluble antigen in the circulation. In one embodiment, the binding polypeptide may reduce the size of the tumor, inhibit the growth of the tumor, and / or prolong the survival time of the cancer-bearing animal. Accordingly, the present disclosure also relates to a method of treating a tumor in such a human or other animal by administering to the human or animal an effective, non-toxic amount of the modified antibody. One of ordinary skill in the art can determine the effective non-toxic amount of a modified binding polypeptide for the purpose of treating a malignant tumor by routine experimentation. For example, the therapeutically effective amount of a modified antibody or fragment thereof is the disease stage (eg, stage I vs. stage IV), age, gender, medical complications (eg, immunosuppressive state or disease), and the body weight of the subject. , And the ability of the modified antibody to elicit the desired response in the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as required by the therapeutic context.

In general, the compositions provided in the present disclosure can be used to prophylactically or therapeutically treat any neoplasm, including antigen markers that allow the targeting of cancerous cells with modified antibodies. ..

VIII. Methods of Administering Modified Antibodies or Fragments thereof The method of preparing the binding polypeptide of the present disclosure and administering it to a subject is well known to those of skill in the art or readily determined by those of skill in the art. The route of administration of the binding polypeptide of the present disclosure may be oral, parenteral, inhalation, or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. Parenteral administration of intravenous, intraarterial, subcutaneous, and intramuscular forms is generally preferred. All of these dosage forms are clearly contemplated to be within the scope of the present disclosure, and the forms for administration are solutions for injection, especially for intravenous or intraarterial injection or infusion. Pharmaceutical compositions suitable for injection usually include a buffer (eg, acetate buffer, phosphate buffer, or citrate buffer), a surfactant (eg, polysorbate), and optionally a stabilizer (eg, human albumin). You may be. However, other methods applicable as taught herein can deliver the modified antibody directly to the site of a population of harmful cells, thereby increasing exposure of the affected tissue to a therapeutic agent. can.

In one embodiment, the binding polypeptide administered is of formula (III) :.
Ab (Gal-C (H) = NQ-CON-X) _x (Gal-Sia-C (H) = NQ-CON-X) _y
Equation (III)
Is a binding polypeptide of
During the ceremony
A) Ab is an antibody defined herein and is an antibody.
B) Q is NH or O,
C) CON is the connection portion specified herein.
D) X is an effector moiety (eg, a therapeutic or diagnostic agent as defined herein).
E) Gal is a component derived from galactose and is a component.
F) Sia is a component derived from sialic acid and is a component.
G) x is 0 to 5,
H) y is 0 to 5,
At least one of x and y is not zero.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and organic esters for injection such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions such as saline and buffering media. The pharmaceutically acceptable carriers in the compositions and methods of the present disclosure are, but are not limited to, 0.01-0.1 M, preferably 0.05 M phosphate buffer, or 0.8% saline solution. Can be mentioned. Other common parenteral vehicles include sodium phosphate solution, dextrose-added Ringer's solution, dextrose and sodium chloride, lactated Ringer, or non-volatile oils. Intravenous vehicles include liquid and nutrient supplements, electrolyte supplements, such as those based on dextrose-added Ringer's solution. For example, preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, and inert gases may also be present. More specifically, pharmaceutical compositions suitable for use for injection are sterile aqueous solutions (if water soluble), or dispersants and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. including. In such cases, the composition must be sterile and have sufficient fluidity to facilitate syringe operability. The composition must be stable under conditions of manufacture and storage and preferably protected from the contaminants of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

Prevention of microbial action can be achieved with a variety of antibacterial and antifungal agents such as parabens, chlorobutanols, phenols, ascorbic acid, thimerosal and the like. In many cases, it is preferable to include an isotonic agent such as sugar, polyalcohol, for example mannitol, sorbitol, or sodium chloride in the composition. Long-term absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

In any case, the sterile injectable solution comprises the effective compound (eg, modified binding polypeptide alone or in combination with other active substances) as appropriate, one of the components listed herein. It can be prepared by placing in the required amount of suitable solvent containing one or a combination and then sterilizing by filtration. Generally, dispersions are prepared by placing the active compound in a sterile vehicle, which contains the basic dispersion medium and other components required from those listed above. For sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying, in which case from a pre-sterile filtered solution containing any additional desired ingredient in addition to the active ingredient. , Get those powders. Preparations for injection are processed and processed according to methods known in the art, filled in containers such as ampoules, bags, bottles, syringes, or vials and sealed under sterile conditions. In addition, the preparations are co-pending U.S., each of which is incorporated herein by reference. S. S. N. 09/259,337 and U.S.A. S. S. N. It may be packaged and sold in the form of a kit such as that described in 09/259,338. Such products have labels or package inserts indicating that the associated composition is useful in treating subjects who are suffering from or have a predisposition to an autoimmune or neoplastic disorder. It is preferable to have.

Effective doses of the compositions of the present disclosure for treating the conditions described above include the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, or any other dose. Drug therapy, and treatment varies depending on many different factors, including prophylactic or therapeutic. Patients are usually human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be determined using conventional methods known to those of skill in the art to optimize safety and efficacy.

For passive immunization with a binding polypeptide, the dose is, for example, about 0.0001 to 100 mg / kg of the body weight of the host, more generally 0.01 to 5 mg / kg (eg 0.02 mg / kg,). It may be in the range of 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, lmg / kg, 2 mg / kg, etc.). For example, the dose may be in the range of 1 mg / kg body weight, or 10 mg / kg body weight, or 1-10 mg / kg, preferably at least 1 mg / kg. Intermediate doses within the above ranges are also considered to be within the scope of the present disclosure. Subjects can receive such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary treatment involves administration in multiple doses over a long period of time, such as at least 6 months. A further exemplary treatment regimen involves administration once every two weeks, once a month, or once every three to six months. An exemplary dosage schedule comprises 1-10 mg / kg or 15 mg / kg daily or 30 mg / kg every other day, or 60 mg / kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the indicated range.

The binding polypeptides of the present disclosure can be administered on multiple occasions. The interval between single doses may be weekly, monthly, or yearly. The intervals may be irregular, as shown by measurements of blood levels in patients with modified binding polypeptides or antigens. Some methods achieve plasma concentrations of 1-1000 μg / ml of the modified binding polypeptide by adjusting the dose, and some methods achieve 25-300 μg / ml. Alternatively, the binding polypeptide may be administered as a sustained-release preparation, in which case it may be administered less frequently. For the antibody, the dose and frequency will vary depending on the half-life of the antibody in the patient. In general, humanized antibodies have the longest half-life, followed by chimeric and non-human antibodies.

The dosage and frequency of administration may be varied depending on whether the treatment is prophylactic or therapeutic. For prophylactic applications, a composition comprising an antibody of the invention or a cocktail thereof may be administered to a patient who is not yet in a diseased state to enhance patient resistance. Such an amount is defined as a "preventive effective dose". In this use, the exact amount will also depend on the patient's health and systemic immunity, but is generally 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose. It is a range. A relatively low dose is administered over a long period of time at relatively infrequent intervals. Some patients will continue to be treated for the rest of their lives. For therapeutic applications, relatively high doses at relatively short intervals (eg, about 1 to 400 mg / kg of antibody per dose, 5 to 25 mg for radioimmunoconjugates are more commonly used. , Cytotoxicity-more doses are used for drug-modified antibodies), but preferably the patient exhibits partial or complete remission of the disease's symptoms until the disease progresses slowly or is terminated. May be required. The patient may then be given a prophylactic regimen.

The binding polypeptides of the present disclosure may optionally be administered in combination with other agents effective in treating a disorder or condition requiring treatment (eg, prophylactic or therapeutic). An effective single-treatment dose (ie, therapeutically effective dose) of the 90Y-labeled modified antibody of the present disclosure ranges from about 5 to about 75 mCi, more preferably between about 10 and about 40 mCi. Effective Treatment of 131I-Modified Antibodies The doses other than a single bone marrow removal range from about 5 to about 70 mCi, more preferably between about 5 and about 40 mCi. A dose for effective treatment single removal of 131I-labeled antibody (ie, which may require autologous bone marrow transplantation) is between about 30 and about 600 mCi, more preferably less than about 50 and less than about 500 mCi. The range between. In combination with the chimeric antibody, the dose of the iodine-131I modified chimeric antibody except for one effective treatment of bone marrow removal ranges from about 5 to about 40 mCi, more preferably less than about 30 mCi. For example, the imaging criteria for the 111In label is typically less than about 5 mCi.

It is emphasized that the binding polypeptide can be administered as described immediately above, but in other embodiments, the binding polypeptide may be administered as a first-line treatment to an otherwise healthy patient. There must be. In such embodiments, the binding polypeptide may be administered to a patient with normal or average red bone marrow accumulation and / or to a patient who has never and is not experienced. Administration of a modified antibody or fragment thereof as used herein in cooperation with or in combination with adjunctive therapy is concomitant, sequential, simultaneous, over a predetermined period of time with the therapeutic and disclosed antibodies. , Or means administration or application at the same time. Those skilled in the art will appreciate that the administration or application of the various components of the concomitant treatment regimen can be timed to enhance the overall effectiveness of the treatment. For example, the chemotherapeutic agent may be administered in a standard, well-known course of treatment followed by the radioimmunoconjugates of the present disclosure within a few weeks. Conversely, cytotoxicity associated with the binding polypeptide may be given intravenously followed by extracorporeal irradiation that is applied topically to the tumor. In yet other embodiments, the modified binding polypeptide may be administered simultaneously with one or more selected chemotherapeutic agents in a single visit. It will be easy for one of ordinary skill in the art (eg, an experienced oncologist) to identify an effective combination treatment regimen without undue experimentation based on the adjuvant therapy of choice and the teachings herein. can.

In this regard, it will be appreciated that the combination of the binding polypeptide and the chemotherapeutic agent can be administered in any order and within any time frame that will bring therapeutic benefits to the patient. That is, the chemotherapeutic agent and the binding polypeptide can be administered in any order or simultaneously. In selected embodiments, the binding polypeptide of the present disclosure is administered to a patient who has previously received chemotherapy. In yet another embodiment, the binding polypeptide and chemotherapeutic treatment are administered substantially simultaneously or in parallel. For example, the patient may be given a binding polypeptide while experiencing the course of chemotherapy. In a preferred embodiment, the modified antibody is administered within 1 year of all chemotherapeutic agents or treatments. In another preferred embodiment, the binding polypeptide is administered within 10, 8, 6, 4, or 2 months of all chemotherapeutic agents or treatments. In yet another preferred embodiment, the binding polypeptide is administered within 4, 3, 2, or 1 week of all chemotherapeutic agents or treatments. In yet another embodiment, the binding polypeptide is administered within 5, 4, 3, 2, or 1 day of the selected chemotherapeutic agent or treatment. It will be further appreciated that the two agents or treatments may be administered to the patient within hours or minutes (ie, substantially simultaneously).

The binding polypeptides of the present disclosure are in vivo in cooperation with or in combination with any chemotherapeutic agent or agent that eliminates, reduces, inhibits, or controls the growth of neoplastic cells. It will be further understood that it can be used (eg, to provide a combination treatment regimen). Exemplary chemotherapeutic agents applicable in the present disclosure include alkylating agents, vinca alkaloids (eg, vincristine and vinblastine), procarbazine, methotrexate, and prednisone. MOPP (mechlorethamine (nitrogen mustard), vincristine (oncovin), procarbazine, and prednisone), which is a combination of the four drugs, is very effective in treating various types of lymphoma and is a preferred practice of the present invention. It includes morphology. In MOPP-resistant patients, ABVD (eg, adriamycin, vinblastine, vinblastine, and dacarbazine), ChIVPP (chlorambusyl, vinblastine, procarbazine, and prednison), CABS (romustin, doxorubicin, breomycin, and streptozotocin), MOPP plus ABVD, MOPP plus ABVD. A combination of plus ABV (doxorubicin, bleomycin, and vinblastine) or BCVPP (carmustin, cyclophosphamide, vinblastine, procarbazine, and prednison) can be used. Arnold S.A. Freedman and Lee M. Nadler, Malignant Lymphomas, HARRISON'S PRINCIPLES OF INTERNAL MEDICINE, pp. 1774-1788 (edited by Kurt J. Isselbacher et al., 13th edition, 1994), and V.I. T. DeVita et al. (1997), as well as references cited herein for standard dosing and planning. These therapies can be used as is, or modified as appropriate for a particular patient, in combination with one or more of the binding polypeptides of the present disclosure described herein.

Additional regimens useful in the context of the present disclosure are single alkylating agents such as cyclophosphamide or chlorambusyl, or CVP (cyclophosphamide, bincristin, and prednison), CHOP (CVP and doxorubicin), C-. MOPP (cyclophosphamide, bincristin, prednison, and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin, and leucovorin), ProMACE-MOPP (prednison, methotrexate, doxorubicin, doxorubicin). Cyclophosphamide, etopocid, and leucovorin, plus standard MOPP), ProMACE-CytaBOM (prednison, doxorbisin, cyclophosphamide, etopocid, citalabine, bleomycin, bincristin, methotrexate, and leucovorin), and MACOP-B (methotrexate). Includes the combined use of doxorubicin, cyclophosphamide, bincristin, fixed doses of predonison, bleomycin, and leucovorin). One of ordinary skill in the art can easily determine standard dosages and plans for each of these regimens. CHOP has also been used in combination with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside, and etoposide. Other applicable chemotherapeutic agents include, but are not limited to, 2-chlorodeoxyadenosine (2-CDA), 2'-deoxycoformycin, and fludarabine.

Salvage therapy is used for patients with moderate and high-grade NHL who are unable to achieve remission or recurrence. Salvage therapy uses drugs such as cytosine arabinoside, carboplatin, cisplatin, etoposide, and ifosfamide that are administered alone or in combination. The following protocols are often used for certain neoplastic disorders with recurrent or fast-growing forms: IMVP-16 (iphosphamide, methotrexate, and etoposide), each at a well-known dosing rate and schedule. , MIME (methyl-gag, iphosphamide, methotrexate, and etoposide), DHAP (dexamethasone, high-dose cytarabine, and cisplatin), ESHAP (etoposide, methylpredonizolone, HD cytarabine, cisplatin), CEPP (B) (cyclophosphamide) , Etoposide, procarbazine, prednison, and bleomycin), and CAMP (lomustine, mitoxanthrone, cytarabine, and prednison).

The amount of chemotherapeutic agent used in combination with the modified antibodies of the present disclosure may vary from subject to subject and can be administered according to what is known in the art. See, for example, Bruce A, Chabner et al., Antineoplastic Agents, GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, pp. 1233-1287 (Jol.

As discussed above, the binding polypeptides, immunoreactive fragments, or recombinants thereof of the present disclosure may be administered in pharmaceutically effective amounts for treating mammalian disorders in vivo. In this regard, the binding polypeptides of the present disclosure are formulated to facilitate administration of the active agent and promote its stability.

The pharmaceutical composition according to the present disclosure preferably comprises a pharmaceutically acceptable, non-toxic, sterile carrier such as a physiological saline solution, a non-toxic buffer, a preservative and the like. For the purposes of this application, a pharmaceutically effective amount of a modified binding polypeptide, immunoreactive fragment, or recombinant thereof conjugated or unconjugated to a therapeutic agent is effective against the antigen. It is said to mean an amount sufficient to achieve binding, and an amount sufficient to achieve an advantage, for example, to alleviate the symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, it is preferred that the modified binding polypeptide can interact with selected immunoreactive antigens on neoplasms or immunoreactive cells, resulting in increased killing of these cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

To maintain the scope of the invention, the binding polypeptides of the present disclosure may be administered to humans or other animals in an amount sufficient to provide a therapeutic or prophylactic effect according to the treatment method described above. .. Such humans or others in conventional dosage forms prepared by combining the binding polypeptides of the present disclosure with conventional pharmaceutically acceptable carriers or diluents according to known techniques. Can be administered to animals. Those skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent will depend on the amount of active ingredient to be combined, the route of administration, and other well-known variables. .. Those skilled in the art will further appreciate that cocktails containing one or more of the binding polypeptides described in the present disclosure may prove to be particularly effective.

IX. Expression of Binding Polypeptides In one aspect, the invention provides a polynucleotide encoding a binding polypeptide disclosed herein. Also provided is a method of making a binding polypeptide comprising expressing these polynucleotides.

The polynucleotide encoding the binding polypeptide disclosed herein is inserted into an expression vector for introduction into a host cell that can be used to produce the desired amount of the claimed antibody or fragment thereof. Is typically done. Accordingly, in certain embodiments, the present invention provides the polynucleotides disclosed herein, as well as expression vectors comprising these vectors and host cells containing the polynucleotides.

As used herein, the term "vector" or "expression vector" is used in accordance with the present invention as a vehicle for introduction and expression in a desired gene in a cell, for the purposes of the present specification and claims. It is used to mean a vector. As known to those of skill in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. In general, a vector applicable in the present invention is a selectable marker, a restriction site suitable for facilitating cloning of the desired gene, the ability to invade and / or replicate in eukaryotic or prokaryotic cells. including.

A large number of expression vector systems can be used for the purposes of the present invention. For example, a class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. do. Others involve the use of polycistronic systems with internal ribosome binding sites. In addition, cells that incorporate DNA into their chromosomes can be selected by introducing one or more markers that allow the selection of transfected host cells. The marker may confer prototrophic, biocide resistant (eg, antibiotic resistant), or resistance to heavy metals such as copper to auxotrophic hosts. The gene of the selectable marker may be directly linked to the DNA sequence to be expressed, or may be introduced into the same cell by co-transformation. Additional factors may be required for optimal synthesis of mRNA. These elements may include a signal sequence, a splice signal, as well as a transcriptional promoter, enhancer, and termination signal. In a particularly preferred embodiment, as discussed above, the cloned variable region gene is synthetically inserted into the expression vector together with the heavy and light chain constant region genes (preferably human).

In another preferred embodiment, the binding polypeptide of the invention may be expressed using a polycistronic construct. In such an expression system, multiple gene products of interest, such as the heavy and light chains of an antibody, may be generated from a single polycistronic construct. These systems are advantageous to use internal ribosome entry sites (IRES) to provide relatively high levels of the polypeptides of the invention in host eukaryotic cells. Applicable IRES sequences are disclosed in US Pat. No. 6,193,980, which is incorporated herein by reference. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of polypeptides disclosed in this application.

More generally, after preparing a vector or DNA sequence encoding an antibody, or a fragment thereof, an expression vector may be introduced into a suitable host cell. That is, the host cell may be transformed. The introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those of skill in the art. Such techniques include, but are not limited to, transfection (including electrophoresis and electroporation), plasma fusion, calcium phosphate precipitation, cell fusion with coated DNA, microinjection, and intact virus. Infection. Ridgway, A.M. A. G. , "Mammalian Expression Vectors", Chapter 24. 2, 470-472, Vectors, Rodriguez and Denhardt eds. (Butterworths, Boston, Mass., 1988). Most preferably, the plasmid is introduced into the host by electroporation. Transformed cells are grown under conditions suitable for light and heavy chain formation and assayed for heavy and / or light chain protein synthesis. An exemplary assay technique is the enzyme-bound immunoadsorption assay (E).
LISA), radioimmunoassay (RIA), or fluorescence activated cell sorter analysis (FACS), immunohistochemistry and the like.

As used herein, the term "transformation" is used broadly to mean the introduction of DNA into a recipient host cell that alters the genotype and results in a change in the recipient cell.

In the same context, "host cell" means a cell transformed with a vector encoding at least one heterologous gene constructed using recombinant DNA technology. The terms "cell" and "cell culture" are used interchangeably in the description of the process for isolating a polypeptide from a recombinant host, and unless otherwise specified, the source of the antibody. Means. In other words, recovery of the polypeptide from "cells" can mean either recovery from centrifuge whole cells or recovery from cell cultures containing both media and suspended cells.

In one embodiment, the host cell line used for antibody expression is of mammalian origin and one of ordinary skill in the art would determine the particular host cell line optimal for the desired gene product to be expressed therein. Can be done. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary system, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney system), COS ( CVI derivative with SV40 T antigen), R1610 (Chinese hamster fibroblasts) BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney system), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells) ), RAJI (human lymph cells), 293 (human kidneys). In one embodiment, the cell line results in altered glycosylation of the antibody expressed therein, eg, afucosylation (eg, PER.C6.RTM. (Crucell) or FUT8-knockout CHO). Cell line (Potellient.RTM. Cell) (Biowa, Vaccineson, NJ)). In one embodiment, NS0 cells can be used. CHO cells are particularly preferred. Host cell lines are typically available from commercial services, the American Cell Lineage Preservation Agency, or published literature.

In vitro production can also be scaled up to obtain large amounts of the desired peptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and are in uniform suspension cultures such as in airlift reactors or continuous stirring reactors, or in hollow fibers, microcapsules. , Includes cell cultures immobilized or captured on agarose microbeads, or on ceramic cartridges and the like. If necessary and / or optionally, the solution of the polypeptide is subjected to conventional chromatography methods such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immunity) affinity chromatography. Can be purified by.

The genes encoding the binding polypeptides of the invention can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard, it will be appreciated that a variety of unicellular non-mammalian microorganisms, such as bacteria, that can be grown in culture or during fermentation, can also be transformed. Bacteria are susceptible to transformation, such as Enterobacteriaceae species, such as Escherichia coli or Salmonella strains, Bacillaceae, such as Bacillus subtilis, Pneumococcus. , Streptococcus, and Haem
Includes types of opilus influenzae). It will be further understood that the polypeptide may be part of inclusion bodies when expressed in bacteria. The polypeptide must be isolated, purified and then assembled into a functional molecule.

In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, the common baker's yeast, is the most commonly used eukaryotic microorganism, but a number of other strains are also commonly available. Expression in Saccharomyces cerevisiae includes plasmid YRp7, eg (Stinchcomb et al., Nature, Vol. 282, p. 39 (1979); Kingsman et al., Gene, Vol. 7, p. 141 (1979); Tschemper et al., Genee, vol. 10, 157 pages (1980)) are commonly used. This plasmid already contains the TRP1 gene, which is a selectable marker for yeast mutants that are incapable of growing in tryptophan, eg, ATCC No. 44076 or PEP4-1 (Jones, Genetics, Vol. 85, p. 12 (1977)). The presence of trp1 disruption as a characteristic of the yeast host cell genome provides an effective environment for detecting transformation due to proliferation in the absence of tryptophan.

The present invention will be further described by the following examples, but the following examples should not be construed as further limitations. Sequence listings, figures, and the content of all references, patents, and published patent applications cited throughout this application shall be incorporated herein by reference in particular.

Designing, Preparing, and Characterizing Variants of 2C3 Anti-CD-52 Highly Glycosylated Antibodies Bulky groups are associated with interacting interfaces (eg, antibodies, eg, antibodies) to regulate the effector function of the antibody by altering its interaction with the FcγR. For the purpose of adding to FcRn binding sites for regulating pharmacokinetics of, or for conjugation of effector moieties, including but not limited to drugs, toxins, cytotoxic agents, and radionuclear species. To introduce chemical modification of the subsequence of the cross-linking site, multiple hyperglycosylated mutations were designed on the heavy chain of 2C3, an anti-CD-52 antibody. The hyperglycosylated 2C3 variants are listed in Table 3.

1A. Preparation of hyperglycosylated variants of 2C3 anti-CD-52 antibody A114N mutations designed based on Kabat's numbering system were introduced into the CH1 domain of 2C3 by mutagenic PCR. To generate a full-length antibody, VH domain plus mutated A114N residues were inserted into the pENTR-LIC-IgG1 vector encoding antibody CH domains 1-3 by ligation-independent cloning (LIC). All other mutations were introduced onto pENTR-LIC-IgG1 by site-directed mutagenesis with a QuickChange site-directed mutagensis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). VH of WT2C3 was cloned by LIC into a mutated vector. The full-length mutant was cloned into a pCEP4 (-E + I) Best expression vector by Gateway cloning. Fc mutations were designed based on the EU numbering system. Mutations were confirmed by DNA sequencing. The amino acid sequences of the heavy and light chains of WT2C3, as well as the amino acid sequences of the mutated 2C3 heavy chains, are shown in Table 4. The mutated amino acids are highlighted in gray and the consensus glycosylation target sites created by the mutation are underlined.

Mutants and WT controls were transfected into HEK2933-EBNA cells in 6-well plate format. Analysis by SDS-PAGE and Western blot, as shown in FIG. 9, found that the expression level was approximately 0.1 μg / ml. In addition, expression of mutants in conditioned medium was also measured by capture of protein A on Biacore. Concentrations were determined using dissociation response 6 minutes after infusion into immobilized protein A. CHO-generated WT2C3, serially diluted from 90 μg / mL to 1.5 ng / mL in medium, was used as the standard curve. Concentrations were calculated up to approximately 0.2 μg / mL by calibration curve using 4-parameter adaptation. As shown in FIG. 9, the relative expression level was low, which was in good agreement with the results of Western blotting.

1B. Verification of hyperglycosylation To determine if additional glycosylation sites were introduced by mutation, 2C3 variants and wild-type proteins were treated with the universal deglycosylation enzyme PNGaseF and sampled. Was analyzed by SDS-PAGE and Western blot. As shown in FIG. 10, only the A114N mutant had an apparent increase in molecular weight, indicating the presence of additional N-linked carbohydrates.

Small-scale antibody preparations were generated and 2C3 variants were purified to further validate the introduction of glycosylation sites. As shown in FIG. 11, it was confirmed that only the A114N mutant had the introduction of a further glycosylation site.

1C. Binding Properties of 2C3 Anti-CD-52 Mutant Biacore was used to compare the binding properties of purified proteins. Mice and human FcRn-HPC4 purified by SEC were immobilized on a CM5 chip by amine coupling. Each antibody was diluted to 200, 50, and 10 nM and injected onto immobilized Fc receptors. Campath, CHO-generated WT 2C3, and DEPC-treated Campath were included as positive and negative controls. As shown in FIG. 13, the Y436 mutant showed a approximately 2-fold reduction in binding to human FcRn. Interestingly, the binding of this mutant to mouse FcRn was unaffected. None of the other 2C3 variants had any significant effect on human or mouse FcRn binding.

Using Biacore, the antigen binding properties of the purified protein were compared using the Biacore binding assay of 741 which is the CD-52 peptide. The CD-52 peptide 741 and the target peptide 777 were immobilized on a CM5 chip. Antibodies were serially diluted 2-fold from 60 to 0.2 nM in HBS-EP, injected twice for 3 minutes each, and then separated by 5 in buffer at a flow rate of 50 μL / min. GLD52 lot 17200-084 was included as a control. The surface was regenerated with one pulse of 40 mM HCl. A 1: 1 coupling model was used to fit curves from 7.5 to 0.2 nM. As shown in FIG. 16, the CD-52 binding affinity of the A114N mutant was slightly lower, and the affinity of the NGT mutant was slightly higher than the remaining mutants in this assay. The Biacore binding assay for the CD-52 peptide 741 was repeated with proteins purified from a larger scale preparation (prep). As shown in FIG. 17, the A114N mutant showed CD-52 peptide binding comparable to 2C3 in WT.

1D. Characterizing the Charge of the A114N Mutant Isoelectric focusing (IEF) was performed to characterize the charge of the 2C3 mutant. The purified protein was run on an acrylamide (IPG) gel with an immobilized pH gradient (pH 3-10). As shown in FIG. 18A, A114N was found to have a more negative charge, probably due to sialic acid residues. Intact MS data confirmed the complex structure of sialic acid on the A114N mutant. In contrast, WT 2C3 has been shown to have G0F and G1F as the predominant glycosylation species (FIGS. 18C and 18D, respectively).

Preparation of hyperglycosylated variants in the backbone of some antibodies In addition to the 2C3 anti-CD-52 antibody, A114N mutations have been applied to some of the other antibody backbones and heavy chain variability unrelated to the unique hyperglycosylated site. Confirmed that it can be introduced in the domain array. Highly glycosylated anti-TEM1, anti-FAP, and anti-Her2 variants are listed in Table 5.

2A. Preparation of hyperglycosylated variants of anti-TEM1 and anti-FAP antibodies The A114N mutation, named based on Kabat's numbering system, was introduced by mutagenic PCR into the CH1 domain of anti-TEM1 and anti-FAP. Mutant VH plus residues 114 were inserted into the pENTR-LIC-IgG1 vector encoding CH domains 1-3 of the antibody by ligation-independent cloning (LIC) to generate a full-length antibody. The full-length mutant was then cloned into the pCEP4 (-E + I) Dest expression vector by Gateway cloning. Mutations were confirmed by DNA sequencing. The anti-TEM1 wild-type amino acid sequences, as well as the mutated heavy and light chain amino acid sequences, are shown in Table 6. The mutated amino acids are highlighted in gray and the glycosylation target sites of the consensus created by the mutation are underlined.

Mutant and wild-type controls were transfected into HEK298-EBNA cells in triple flask format and purified on a HiTrap protein A column (GE Healthcare Biosciences, Pittsburgh, PA, USA). Analysis by NanoDrop spectrophotometric A280 revealed that the expression of anti-FAP A114N and anti-FAP A114C was about 3 μg / ml and about 1 μg / ml, respectively. The expression of A114N for anti-TEM1 was about 0.04 μg / ml.

2B. Verification of hyperglycosylation To confirm that additional glycosylation sites have been introduced into the A114N mutant, proteins purified from the A114N mutant, along with wild-type control proteins, on reducing SDS-PAGE. analyzed. One additional glycosylation site adds 2000-3000 daltons to the molecular weight of the heavy chain. As shown in FIG. 20, SDS-PAGE increased the apparent molecular weight of the heavy chain bands of the anti-FAP and anti-TEM1 A114N mutants, and additional glycosylation sites were successfully introduced into both antibodies. It is pointed out that it was consistent with.

2C. Preparation of hyperglycosylated mutants of anti-Her2 antibody Her-2 A114N, Her-2 A114N / NNAS, and WT Her-
Two antibodies were made by ligation-independent cloning. The VH domain of Herceptin was synthesized and PCR amplified with either two sets of LIC-compatible primers, either WT or having the A114N mutation. To obtain a full-length antibody, the amplified VH insertion fragment (WT or A114N) was placed in two pENTR vectors encoding the CH1-3 domain, the WT of pENTR-LIC-IgG1 and the NNAS of pENTR-LIC-IgG1. Clone to, resulting in three full-length variants (A114N, NNAS, A114N / NNAS) and WT controls as entry clones on pENTR. These variants were cloned into the pCEP4 (-E + I) Best expression vector by Gateway cloning. Mutations were confirmed by DNA sequencing. The wild-type amino acid sequences of anti-Her-2, as well as the mutated heavy and light chain amino acid sequences are shown in Table 7. The mutated amino acids are highlighted in gray and the consensus glycosylation target sites created by the mutation are underlined.

2D. Expression of hyperglycosylated variants of A114N anti-Her2 antibody A114N anti-Her2 and wild-type constructs were prepared with Lipofectamine-2000 (reagent to DNA ratio 2.5: 1) and XtremeGene HP (reagent to DNA ratio 3: 1). Then, the cells were transfected into HEK2933-EBNA cells in 12 triple flasks. Octet measurements of aliquots from conditioned medium (CM) on day 3 showed that protein expression was constant across 6 flasks for both Lipofectamine 2000 and XtremeGene HP. As shown in Table 8, the overall transfection efficiency was 30% higher on XtremeGene HP. The conditioned medium collected on day 3 was pooled together for both transfection conditions and purified by a protein A column. Measurements with Octet showed that the antibody in the serum-containing simulated medium was 1.8 μg / ml, whereas it was 0 μg / ml in the serum-free simulated medium.

The conditioned medium from day 6 was collected and purified separately for each transfection condition. Both eluents were buffered separately in PBS, pH 7.2 and concentrated approximately 15-fold using an Amicon-4 (cutoff 50 kD) column. The CM on the 6th day showed a higher expression level than the CM on the 3rd day. As shown in Table 8, a total of 3 mg of Herceptin A114N 15.59 mg / ml (from lipofectamine transfection) and 6 mg of Herceptin A114N 16.86 mg / ml (from XtremeGene HP transfection), antibody-drug conjugation, etc. , Generated from day 6 conditioned medium for further downstream application.

2E. SDS-PAGE and HIC analysis of A114N anti-Her2 variants Prior to conjugates, purified A114N Herceptin was characterized by SDS-PAGE and HIC (hydrophobic interaction chromatography). As shown in FIG. 21, the quality of purified A114N Herceptin was determined to be suitable for further downstream application.

2F. Conjugation for modified glycosylation The following was demonstrated: a) the glycosylation site was introduced at the Kabat 114 position on anti-TEM1, b) the A114N variant was weighted by reducing SDS-PAGE. It had hyperglycosylation on the chain, and c) the A114 hyperglycosylated variant had a complex carbohydrate structure containing terminal sialic acid and galactose ideal for SAM and GAM conjugation by Intact LC / MS. Had. To confirm that the modified glycosylation site was suitable for conjugation, anti-TEM1 A114N was conjugated with 5 kDa PEG by an aminooxy chemical reaction. As shown in FIG. 22, PEG was successfully conjugated to anti-TEM1 A114N by aminooxy ligation. This variant was also successfully prepared (not shown) on the 2C3 backbone of anti-FAP and anti-CD-52. These data demonstrate that the glycosylation site of N114 is useful for conjugation of effector moieties.

Production of Fc variants of S298N / Y300S A modified Fc variant was designed and produced in which a new glycosylation site was introduced at the Ser298 position in the EU next to the naturally occurring Asn297 site. Glycosylation of Asn297 was either maintained or removed by mutation. The results of the mutation and desired glycosylation are shown in Table 9.

3A. Preparation of Modified Glycosylation Variants of H66αβ-TCR Antibody The heavy chain of αβT cell receptor antibody clone # 66 was mutated by Quikchange using the pENTR_LIC_IgG1 template. The VH domain of HEBE1 Δab IgG1 # 66 was amplified with LIC primers and then cloned by LIC into mutant or wild-type pENTR_LIC_IgG1 to generate full-length mutant or wild-type antibodies. Subcloning was validated with the DraIII / XhoI double digest and inserts of approximately 1250 bp size were generated in successful clones. These full-length mutants were then cloned into the expression vector pCEP4 (-E + I) Dest by Gateway cloning. Mutations were confirmed by DNA sequencing. Table 10 shows the amino acid sequences of the WT H66 anti-αβ TCR heavy chain and light chain, as well as the amino acid sequences of the mutated H66 heavy chain. The mutated amino acids are highlighted in gray and the consensus glycosylation target sites created by the mutation are underlined.

Constructs of mutants, wild-type, and two non-glycosylated controls (HEBE1 Agly IgG4 and HEBE1 Δab IgG1 in pCEP4) were transfected into HEK2933-EBNA cells in triple flasks for expression. Protein was purified from 160 ml of conditioned medium (CM) with 1 ml of HiTrap protein A column (GE) using a multi-channel peristaltic pump. 5 micrograms of each supernatant obtained were analyzed on 4-20% Tris-Glycine reducing and non-reducing SDS-PAGE gels (see Figure 2). Heavy chains of non-glycosylated variants (N297Q, T299A, and Agly controls) migrate farther (arrows), consistent with the loss of glycans in these antibodies. However, the heavy chains of the modified glycosylation antibodies (NSY, STY, SY, Δab, and wt controls, arrows) migrated similarly to wild-type controls. This result was consistent with the presence of a modified glycosylation site at position EU298. SEC-HPLC analysis pointed out that all variants are expressed as monomers.

3B. Glycosylation analysis by LC-MS The modified Fc variant of H66 IgG1 was partially reduced with 20 mM DTT at 37 ° C. for 30 minutes. Samples were then analyzed by capillary LC / MS on an Agent 1100 capillary HPLC system coupled with a QSTARqq TOF hybrid system (Applied Biosystems). Protein reconstruction by Bayesian theory with baseline correction and computer modeling in Analyst QS1.1 (Applied Computer) were used for data analysis. In the H66 antibody variant of S298N / T299A / Y300S, one glycosylation site was observed at amino acid 298, and bifurcated and trifurcated complex glycans were detected as the major species alongside G0F, G1F, and G2F. (See Figure 34). This modified glycosylation profile is consistent with the transglycosylation of N298 instead of the wild-type N297 glycosylation site.

3C. Binding Properties of αβTCR Antibody Variants to Human FcγRIIIa and FcγRI Using Biacore Biacore was used to evaluate the binding to recombinant human FcγRIIIa (V158 and F158) and FcγRI. All four flow cells of the CM5 chip were immobilized with anti-HPC4 antibody by the standard amine coupling procedure provided by Biacore. The anti-HPC4 antibody was diluted to 50 μg / mL in 10 mM sodium acetate pH 5.0 for the coupling reaction and injected at 5 μL / min for 25 minutes. Approximately 12000 RU of antibody was immobilized on the chip surface. Recombinant human FcγRIIIa-V158 and Fc
γRIIIa-F158 was diluted to 0.6 μg / mL in binding buffer (HBS-P containing 1 mM CaCl ₂ ) and injected into flow cells 2 and 4 at 5 μL / min for 3 minutes, respectively, 300-400 RU on anti-HPC4 chip. The receptor was captured. To distinguish between low binders, 3 times more rhFcγRIIIa than normally used in this assay was captured on the anti-HPC4 surface. Flow cells 1 and 3 were used as reference controls. Each antibody was diluted to 200 nM in binding buffer, injected onto all four flow cells for 4 minutes and subsequently dissociated in buffer for 5 minutes. The surface was regenerated with 10 mM EDTA in HBS-EP buffer at 20 μL / min for 3 minutes. The results of these experiments are shown in FIG.

Biacore was also used here to compare FcγRI binding. The anti-tetraHis antibody was buffer exchanged to 10 mM sodium acetate pH 4.0 using a Zeba Deserting column and diluted to 25 μg / mL in acetate buffer for amino coupling. Two flow cells of the CM5 chip were injected at 5 μL / min for 20 minutes and then immobilized with approximately 9000 RU of anti-tetraHis antibody. To compare the sample with weak binding, 10 times more FcγRI was captured on the anti-tetraHis surface as in the previous experiment. Recombinant human FcγRI was diluted 10 μg / mL in HBS-EP binding buffer and injected into Flowcell 2 at 5 μL / min for 1 minute to capture approximately 1000 RU receptors on an anti-tetraHis chip. A single concentration of 100 nM antibody was injected onto the surface of the captured receptor and control at 30 μL / min for 3 minutes. The dissociation was subsequently monitored for 3 minutes. Then, 10 mM glycine pH 2.5 was injected twice at 20 μL / min for 30 seconds to regenerate the surface. The results of these experiments are shown in FIG.

These results demonstrate a significant reduction in the binding of sugar-modified mutants to FcγRIIIa or FcγRI. In particular, the binding of H66 to both receptors of S298N / T299A / Y300S was almost completely disrupted. This variant was selected for further analysis.

3D. Characterizing Stability Using Circular Dichroism Monitor the stability of variants of the S298N / T299A / Y300S antibody with CD signals at 216 nm and 222 nm as elevated temperatures resulting in antibody unfolding (denaturation). , Monitored by far UV CD thermal melting experiments.

The temperature was controlled by a thermoelectric Peltier (JASCO model AWC100) and increased from 25-89 ° C to a rate of 1 ° C / min. CD spectra were collected by Jasco 815 spectrophotometric counting at a protein concentration of approximately 0.5 mg / mL in PBS buffer in quartz cuvette (Hellma, Inc) with an optical path length of 10 mm. The scan speed was 50 nm / min and the data pitch was 0.5 nm. The sensitivity was set to medium and a bandwidth of 2.5 nm was used. CD signals and HT potentials were recovered from 210-260 nm with a data interval of 0.5 nm and a temperature interval of 1 ° C., and each sample was subjected to four overlapping scans. The results demonstrate that both the H66 variants of Delta AB and the H66 variants of S298N / T299A / Y300S show similar thermal behavior and have approximately the same starting temperature (around 63 ° C.) for degradation (around 63 ° C.). FIG. 35) further suggests that they have comparable stability.

Functional analysis of Fc-modified mutants Fc-modified mutants were evaluated by PBMC proliferation assay and cytokine release assay. In the PBMC proliferation assay, human PBMCs were cultured with increased concentrations of therapeutic antibody for 72 hours, ^3H -thymidine was added, and cells were harvested after 18 hours. In the T cell depletion / cytokine release assay, human PBMCs were cultured with increased concentrations of therapeutic antibody and analyzed daily for cell number and viability up to day 7 (Vi-Cell, Beckman Coulter). Cell supernatants were also collected, stored at −20 ° C. and analyzed on a cytokine panel (Bio-Rad) with 8 components (8 compartments).

PBMCs of normal donors were thawed and treated under the following conditions (all in medium containing complement): untreated; BMA031, moIgG2b 10 μg / ml; OKT3, moIgG2a 10 μg / ml; H66, huIgG1 Delta AB 10 μg / ml. 1, 1 μg / ml, and 0.1 μg / ml; H66, huIgG1 S298N / T299A / Y300S 10 μg / ml, 1 μg / ml, and 0.1 μg / ml.

Cytokines were collected for BioPlex analysis on days 2 (D2) and 4 (D4) (IL2, IL4, IL6, IL8, IL10, GM-CSF, IFNg, TNFa). To D4, cells were stained for CD4, CD8, CD25, and abTCR expression.

The results shown in FIGS. 5-8 show that S298N / T299A / Y300S of H66 behaved similarly to Delta AB of H66 in all cell-based assays performed, with respect to minimal activation of T cells by CD25 expression, abTCR. It demonstrates binding (with slightly different kinetics to Delta AB), as well as minimal release of cytokines at both D2 and D4 time points. The S298N / T299A / Y300S mutant thus eliminated effector function as effectively as the Delta AB mutation.

Preparation and characterization of the modified Fc variant in the backbone of the anti-CD52 antibody In addition to the H66 anti-αβ TCR antibody, the S298N / Y300S mutation was also modified in the backbone of the anti-CD52 antibody (clone 2C3). This variant was then tested to determine if the observed regulation of effector function in the S298N / Y300S H66 anti-αTCR antibody was consistent with the backbone of another antibody.

5A. Preparation of Glycosylated Variant Modified by 2C3 Anti-CD52 Antibody First, 2C3 variant DNA of S298N / Y300S was prepared by quick change mutagenesis using pENTR_LIC_IgG1, and 2C3 VH of WT was cloned by LIC into the mutated vector. .. The full-length mutant was cloned into the pCEP4 (-E + I) Dest expression vector using the Gateway technique. The mutations were subsequently confirmed by DNA sequencing and the sequences were listed in Table 11. The mutant was then transfected into HEK2933-EBNA cells in 6-well plate format and the protein was purified from conditioned medium. Anti-CD52 2C3 wild-type antibody was produced in parallel as a control. Expression levels were found to be 0.1 μg / mL using SD-PAGE and Western blot analysis (FIG. 9A). Expression of variants in neat conditioned medium was also measured by capture of protein A on Biacore. The concentration was determined using the dissociation response after injecting the immobilized protein A for 6 minutes. CHO-generated WT2C3 was serially diluted from 90 μg / mL to 1.5 ng / mL in medium and used as a standard curve. The concentration was calculated to be within approximately 0.2 μg / mL by a calibration curve using a four-parameter fit. Relative expression levels were low and were in good agreement with Western blot data (FIG. 9B).

5B. Glycosylation analysis with PNGaseF To evaluate the further glycosylation site introduced by the mutation, the enriched S298N / Y300S mutant was deglycosylated with PNGaseF. It was pointed out that no further carbohydrates were present, as no apparent change in molecular weight was demonstrated (Fig. 10). Small scale preparations were made to purify these variants for further characterization and the results reaffirmed the absence of additional carbohydrates on the S298N / Y300S variants (FIG. 11).

5C. Biacore-based 2C3 anti-CD-52 antibody variant binding to human FcγRIIIa Biacore was also used to characterize antigen-binding, FcγRIII, and purified antibody binding properties (FIGS. 12, 13, and 14). Please refer to). The 2C3 variant of S298N / Y300S binds tightly to the CD52 peptide and the binding sensorgram is indistinguishable from wild-type controls, demonstrating that this mutation does not affect antigen binding (Figure). 12A).

The FcγRIII receptor (Val158) was used in the binding test to assay for Fc effector function. Mutant and wild-type control antibodies were diluted to 200 nM and
It was injected into FcγRIIIa captured by HPC4-tag. FcγRIII binding was almost undetectable for the S298N / Y300S mutant, indicating a loss of effector function due to this variant (FIGS. 12B and 14A). The FcγRIII receptor (Phe158) was also used in the binding test to further assay for Fc effector function. Mutant and wild-type control antibodies were diluted to 200 nM and injected into FcγRIIIa captured by HPC4-tag. Binding of FcγRIII was almost undetectable for the S298N / Y300S mutant, indicating a loss of effector function in the Phe158 variant (FIG. 14B). Finally, Biacore was used to compare the FcRn binding properties of the purified proteins. Mouse and SEC-purified human FcRn-HPC4 were immobilized on CM5 chips by amine coupling. Each antibody was diluted to 200, 50, and 10 nM and injected onto the receptor. Campath, CHO-generated WT2C3, and DEPC-treated Campath were included as positive and negative controls. These data indicate that the mutant may bind to both human and mouse FcRn receptors with the same affinity as wild-type antibody controls and may alter cyclic half-life or other pharmacokinetic properties. It indicates that there is no such thing (see FIGS. 12C, 13A, and B). Therefore, the S298N / Y300S mutation is generally applicable to antibodies to reduce or eliminate unwanted Fc effector function, such as by the involvement of human Fcγ receptors.

Detection of Circulating Immune Complexes in S298N / Y300S Mutants Detection of circulating immune complexes was also investigated using the C1q binding assay against S298N / Y300S mutants and WT controls. Highly binding Costar 96-well plates were coated overnight at 4 ° C. with 100 μl of 2C3Ab diluted 2-fold in a coating buffer (0.1 M NaCHO ₃ , pH 9.2) at concentrations ranging from 10 to 0.001 μg / ml. ELISA analysis showed that C1q binding was reduced in the S298N / Y300S mutant compared to WT (FIG. 15A). Binding of anti-FabAb to the coated 2C3Ab confirmed an equal coating of wells (FIG. 15B).

Separation and Analysis of S298N / Y300S Mutants Using Isoelectric Focusing An isoelectric focusing (IEF) gel with a pH of 3-10 was run to characterize the S298N / Y300S mutants. S298N / Y300S was found to have a more negative charge and is therefore likely to be a sialic acid molecule (FIG. 18A). Both the S298N / Y300S mutant and 2C3 of WT were shown by intact MS to have G0F and G1F as the predominant glycosylation species (FIGS. 18B and D, respectively).

Antigen Binding Affinities of S298N / Y300S Biacore of 2C3Ab and S298N / Y300S variants of WT anti-CD-52 prepared and purified from both small scale (FIG. 16) and large scale (FIG. 17) expression. Antigen binding affinities were compared. CM5 chips immobilized with the CD52 peptide 741 and the control peptide 777 were obtained. Antibodies were serially diluted 60 to 0.2 nM in HBS-EP, then injected over the chip surface for 3 minutes and subsequently dissociated in buffer at a flow rate of 50 μl / min for 5 minutes. The surface was then regenerated with a pulse of 40 mM HCl. These analyzes were performed twice each, demonstrating that the S298N / Y300S mutant and the WT 2C3 antibody show comparable CD52 peptide binding.

To screen for antibodies produced during small-scale transfections, a media screening platform was designed and tested for pre-purification functional binding properties. These tests were performed using Octet (FIG. 19A) to determine the concentration and a protein A biosensor and GLD52 standard curve were used. Samples were diluted to 7.5 and 2 nM in HBS-Ep for comparison of CD52 binding with Biacore (FIG. 19B). The results of the peptide bond assay showed that both the S298N / Y300S mutant and the WT2C3 antibody had comparable CD52 peptide binding properties. In addition, these analyzes demonstrated that Octet and Biacore work well to predict antibody-induced antigen binding from small-scale transfections.

Preparation of Glycosylated Variants Modified by S298N / Y300S, S298N / T299A / Y300S, and S297Q / S298N / Y300S in the Backbone of Further Antibodies In addition to anti-αβ-TCR and 2C3 anti-Cd-52 antibodies, S298 / Y300S, The S298N / T299A / Y300S and N297Q / S298N / Y300S mutations were modified in the backbone of other antibodies to confirm that additional tandem glycosylation sites could be introduced into unrelated heavy chain variable domain sequences. The 12G6 and anti-Her2 variants of anti-CD-52 that are alternately glycosylated are listed in Tables 12 and 13.

Production of Modified Antibodies Containing Reactive Glycan Substates To produce antibodies containing glycan moieties that can react with derivatized effector moieties, anti-HER antibodies are first used with glycosyltransferases and associated nucleonucleotide donors. Glycosylated in vitro using. For example, to introduce a sialic acid residue, the donor antibody was first galactosylated with β-galactosyltransferase and then sialylated with α2,6-sialyltransferase according to the method of Kaneko et al. (Kaneko, Y., Nimmerjan, F., and Ravech, JV (2006), Anti-inframmatory activity of immunoglobulin G resulting from Fc sialylation., Science, 313, 670-3). The reaction was carried out with β-galactosyltransferase (50 mU / mg, Sigma) and α2,6-sialyltransferase (5 μg / mg, R & Dsystem) in 50 mM MES buffer (pH 6.5) containing 5 mM MnCl ₂ with the donor nucleotide substrate. It was performed in a one-pot synthesis step with certain UDP-galactose (10 mM) and CMP-sialic acid. The reaction mixture containing 5 mg / ml anti-HER2 antibody was incubated at 37 ° C. for 48 hours. Sialylation was performed with MALDI-TOF MS analysis of overmethylated glycans released from the antibody with PNGaseF, sialic acid content analysis with Dionex HPLC, and with SNA, a lectin specific for α2,6-sialic acid. It was verified using lectin blotting.

MALDI-TOF analysis of the glycans released by the PNGaseF treatment of the sialylated anti-HER2 antibody revealed that the natural glycans were predominantly monosialylated bifurcated in A1F (FIG. 27A) with a small amount of disialylated species. It was pointed out that it was completely rebuilt. Treatment of the antibody with high doses of α2,6-sialyltransferase produces a more uniform population of A1F glycotypes, suggesting that either enzymatic activity or glycan localization can prevent complete sialylation. rice field. The sialic acid content was determined to be approximately 2 mol per mol of antibody, consistent with A1F glycans as the predominant sugar type species (FIG. 27B). Lectin blotting with SAN lectin, an aglutinin of elderberry (Sambucus nigra) specific to α2,6 linked sialic acid, confirmed that sialic acid is present in the α2,6 bound configuration (FIG. 27C). ).

In conclusion, the intrinsically disordered glycans are somewhat heterogeneous, but reconstruction with galactosyl and sialyltransferases yields near-homogeneous antibodies with bifurcated glycans that are monosialylated but completely galactosylated. (A1F). The fact that only one sialic acid was introduced into the two galactose acceptors on each branched glycan means that glycans are often buried in the antibody and therefore can only access one galactose. It may be due to a non-covalent interaction of the glycan with the protein surface.

After verifying the oxidation sialylation of the modified antibody containing the reactive glycan moiety, oxidation of the sialylated anti-HER2 antibody with various concentrations of periodic acid (0.25 to 2 mM) during the manufacturing process. investigated. The sialylated antibody was first buffer exchanged with 25 mM Tris-HCl (pH 7.5) containing 5 mM EDTA, followed by buffer exchange with PBS buffer. The buffered antibody mixture was then applied to a protein A sepharose column pre-equilibriumed with PBS buffer. The column was washed with 15 column volumes of PBS, 15 column volumes of PBS containing 5 mM EDTA, and 30 column volumes of PBS, which was then eluted with 25 mM phosphate buffer (pH 2.9). The eluate was immediately neutralized with dibasic phosphate buffer and the antibody was concentrated using Amicon ultra from Millipore. After purification
The sialylated anti-HER2 antibody was then oxidized with sodium periodate (Sigma) in 100 mM sodium acetate buffer (pH 5.6) for 30 minutes in the dark on ice and reacted with 3% glycerin on ice for 15 minutes. Was quenched. The product was desalted and replaced with 100 mM sodium acetate (pH 5.6) by 5 rounds of extrafiltration on Amicon at 50 kDa. FIG. 28A shows an analysis of the sialic acid content of sialylated antibodies determined with varying amounts of periodate. Complete oxidation of sialic acid residues was achieved at periodate concentrations above 0.5 mM. In fact, a periodate concentration as low as 0.5 mM is sufficient to completely oxidize the introduced sialic acid. Therefore, 1 mM of periodate was chosen for the oxidation of sialylated antibodies for drug conjugates.

Oxidation can have adverse effects on antibody integrity. For example, oxidation of methionine residues, including Met-252 and Met-428 located in the CH3 region of the Fc near the FcRn binding site, may affect FcRn binding, which is crucial for prolonging the serum half-life of the antibody. Known (Wang, W. et al. (2011) Impact of methionine)
oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies. , Mol Immunol, Vol. 48, pp. 860-6). Therefore, in order to investigate the potential side effects of periodic acid oxidation on methionine residues (eg, Met-252) that are decisive for FcRn interactions, the oxidative state of the sialylated antibody was subjected to tryptic peptide digestion. Determined by LC / MS analysis. Analysis revealed approximately 30% oxidation of Met-252 and <10% oxidation of Met-428 after treatment with trastuzumab sialylated with 1 mM periodate. To determine the effect of this degree of methionine oxidation on FcRn binding, the kinetics of FcRn binding to each antibody was evaluated using surface plasmon resonance (BIACORE). This analysis revealed that the state of oxidation correlates with a small loss of FcRn binding (12% and 26% reduction relative to mouse and human FcRn, see FIGS. 28B and 28C, respectively). Approximately 25% reduction in Ka to human FcRn is plasma in human FcRn transgenic mice, especially since a single intact FcRn site on each antibody is sufficient to provide functional and PK benefits. It has been reported to have no effect on the half-life (Wang et al., Ibid.).

In summary, these data show that when periodic acid residues sensitive to periodate are introduced by sialyltransferase treatment, very low concentrations of periodate become available for antibody-FcRn interactions. On the other hand, it is pointed out that the minimum side effects and the uniformity of the antibody evaluated by aggregation (≦ 1%) are brought about. Thus, the use of sialylated antibodies according to the method of the invention provides a wider window of oxidation conditions to be used and reproducibly produces active complex carbohydrates without any effect on serum half-life. become able to.

Galactose in the hyperglycosylated antibody variant can also be specifically oxidized with galactose oxidase to produce an aldehyde group for conjugation. To confirm this approach, A114N anti-TEM1 antibody was concentrated to 13-20 mg / ml and then treated with 20 mU / mg sialidase in PBS at 37 ° C. for 6 hours. The desialylated product was then oxidized with galactose oxidase (“GAO”), first with 5 μg GAO per 1 mg protein overnight at 37 ° C., followed by 2 μg GAO per 1 mg protein and incubated for an additional 5 hours. .. Sodium acetate was added to adjust the pH to 5.6 (0.1 v / v, pH 5.6) and DMSO was added to achieve a final reaction concentration of 16%, added prior to conjugation. The hyperglycosylated variant A114N anti-HER antibody (15 mg / ml) was similarly desialylated with sialidase (20 mU / mg) and 5 μg G per 1 mg protein in a single reaction overnight at 37 ° C.
Oxidized with AO.

Synthesis of Reactive Effector Substituents Candidate drug effector moieties (eg, monomethylauristatin E (MMAE) and drastatin 10 (Dol10)) to facilitate conjugation with the glycotype of the aldehyde derivatized antibody of the invention. Was derivatized with aminooxy-cystamide to contain a functional group that is specifically reactive with the aldehyde (eg, aminooxy-cys).

Briefly, to produce aminooxy-cystamide as a starting material, S-trityl-L-cysteine amide (362 mg, 1 mmol) was added to t-BOC-aminooxyacetic acid N-hydroxysuccinamide ester (289 mg, 1 mmol). 1 mmol) was added to 3 mL of the DMF solution. The reaction was completed after 3 hours, as evidenced by HPLC analysis. Subsequently, the reaction mixture was diluted with 30 ml of dichloromethane and washed with 0.1 M sodium bicarbonate solution (2 x 20 mL), water (2 x 20 mL), and brine (2 x 20 mL). The solution was dried over anhydrous sodium sulfate, filtered, concentrated and dried. To this dried residue was added 3 mL of TFA, followed by 150 μL of triethylsilane. The resulting solution was precipitated from t-butyl methyl ether and this process was repeated 3 times. After filtration, the residue was dried under reduced pressure to give 205 mg of an off-white solid (yield 67%). The compound was used in the next step without further purification.

To produce aminooxy-derivatized MMAE (Aminooxy-Cys-MC-VC-PABC-MMAE), 30.1 mg (0.098 mmol, 2 eq) of aminooxycystamide in 3 mL of DMF was added to MC-VC-PABC. -MMAE (0.049 mmol) 64.6 mg and triethylamine 100 μL were combined. The resulting reaction mixture was stirred at room temperature for 15 minutes and by that time the reaction was complete, according to HPLC analysis. The compound was purified by preparative HPLC to give 45 mg (62%) of the desired product as an off-white solid. Reversed-phase HPLC analysis suggested that the purity of the compound was> 96%. ESI: C73H116N14O18S (MH) ⁺ calculated value 1509.8501; measured value, m / z 1509.8469.

To produce aminooxy-derivatized Dol10 (aminooxy-Cys-MC-VC-PABC-PEG8-Dol10), aminooxycystamide 7.4 mg (0.024 mmol, 3 eq), MC-VC-PABC- 12 mg (0.008 mmol) of PEG8-Dol10 and 30 μL of triethylamine were combined in 3 mL of DMF. The reaction was completed within 15 minutes according to HPLC analysis. Purification by preparative HPLC resulted in 6.2 mg (46%) of the desired product of an off-white solid. Reversed-phase HPLC analysis suggested that the purity of the compound was> 96%. ESI: C80H12
4N16O19S2 (MH) ⁺ calculated value 1678.0664; measured value, m / z 1678.0613.

Sialic acid-mediated (SAM) conjugation of reactive effector moieties After desalting, the drug-linker of Example 11 was oxidized and sialyl of Example 10 at a concentration of 25 mM in 75% DMSO (0.167v / v). Combined with the isomerized antibody, a drug-linker-to-antibody molar ratio of 24: 1 and a final antibody concentration of 5 mg / ml were achieved. The mixture was incubated overnight at room temperature. Non-incorporated drug-linker and any free drug were collected using BioBeads. The product was buffered to histidine-Tween buffer using a PD-10 column and sterile filtered. Determine endotoxin levels and in vivo
ADCs of less than 0.1 EU / mg were achieved in the test.

29A-C show a hydrophobic interaction chromatograph (HIC) of various sialylated antibodies (B11 and G11 of anti-FAP of Example 11 and anti-HER2 antibody) glycoconjugated to AO-MMAE. .. The sialylated HER2 antibody was also conjugated with the drug-linker AO-Cys-MC-VC-PABC-PEG8-Dol10 (FIG. 29D). This analysis revealed that there were predominantly one to two drug conjugates per antibody, with drug-to-antibody ratios (DARs) ranging from 1.3 to 1.9. The prolongation of the retention time of the Do110 glycoconjugate (FIG. 29D) as compared with the MMAE glycoconjugate (FIG. 29C) is considered to be due to the high hydrophobicity of Do110.

LC-MS analysis was also performed with anti-HER antibodies conjugated to two different drug-linkers (AO-MMAE or AO-PEG8-Dol10) on a 30 mg scale. This analysis shows that the post-conjugated DA values are similar at 1.7 and 1.5, which is comparable to the HIC analysis. Size Exclusion Chromatography (SEC) showed very low levels of (1%) aggregates in these conjugates.

Galactose-mediated (GAM) conjugate of the reactive effector portion The antibody was incubated overnight at 25 ° C. with galactose oxidase produced on the A114N anti-TEM1 hyperglycosylated mutant antibody described in Example 11. It was conjugated with an excess of 24 mol of aminooxy-MC-VC-PABC-MMAE drug-linker to give an ADC conjugate of DA1.72. To the galactose oxidase-treated anti-HER antibody prepared as described in Example 11, 1/10 reaction volume of 1M sodium acetate and pH 5.6 was added to adjust the pH to 5.6, and DMSO was adjusted. After making the final concentration 14%, 24 equivalents of aminooxy MC-VC-PABC-MMAE drug linker was added. The reaction was incubated overnight at room temperature. Free drugs and drug-linkers were collected on Biobeads and the product buffer was exchanged by SEC (yield 65%). Product conjugates were analyzed by HIC. As shown in FIG. 30, AO-MMAE was conjugated to approximately 60% of the molecule.

In vitro ADC Cell Proliferation Assay The in vitro activity of the anti-HER and anti-FAP glycoconjugates of the invention is also the corresponding thiol comprising the same drug moiety linked to the hinge region cysteine of the same donor antibody by thiol ligation. Compared to the conjugate. Thiol conjugates contained approximately twice as much drug (DAR) per antibody as complex carbohydrates. Thiol-based conjugation was performed as described by Stefano et al. (Published, Methods in Molecular Biology, 2013). The Her2 + SK-BR-3 and Her2-MDA-MB-231 cell lines were then used to assess the relative efficacy of each ADC. The results of this analysis are shown in Table 15 below.

FIG. 31 shows a comparison of the in vitro efficacy of anti-HER complex carbohydrates and their counterparts, thiol conjugates. Cell viability was exposed to the conjugate to Her2 antigen-expressing (SK-BR-3) cells (FIGS. 31A and C) or non-expressing (MDA-MB-231) cells (FIGS. 31 and D) for 72 hours. It was decided later. The ADC contained either MMAE or PEG8-Do119 linked to a glycan (“glyco”), or cysteine (“thiol”) in the hinge region by conventional chemical reaction. As shown in FIGS. 31A and 31C, EC50 was observed to be approximately 2-fold lower than the thiol conjugate compared to the complex carbohydrate, which is consistent with the 2-fold higher DA in the former than in the latter. No toxicity was observed for Her2 cell lines with any antibody up to 100 μg / ml.

Similar trends are observed in cell proliferation against ADCs prepared with antibodies to tumor antigen (FAP), which are highly expressed by reactive interstitial fibroblasts in colon cancer, pancreatic cancer, and epithelial cancers including breast cancer. (Teicher, BA (2009), Antibody-drug conjugate targets., Curr Cancer Drag Targets, Vol. 9, pp. 982-1004). These conjugates are also prepared by conjugating either the aminooxy MMAE drug-linker or the maleimide MMAE drug-linker to a glycan or thiol group. From these conjugate cell proliferation assays, thiol-conjugated EC ₅₀ was approximately 100-fold more potent against human FAP-transfected CHO cells than the same cells lacking FAP expression, as shown in FIG. However, FIG. 32 shows a comparison of the in vitro efficacy of the anti-FAP B11 complex sugar vs. thiol conjugate. Cell viability was determined after exposure to conjugates for CHO cells transfected with or without FAP antigen. The ADC contained glycan-bound MMAE (“glyco”) or MMAE (“thiol”) bound to cysteine in the hinge region by conventional chemical composition. EC50, which is approximately twice lower for thiols compared to glycoconjugates, is consistent with the relative amount of drug delivered per antibody, assuming similar efficiency for target binding and internalization in antigen-expressing CHO cells. Will be done. In parallel, as previously described, anti-FAP (B11) ADC complex carbohydrates with _DAT1.5 were assayed and showed approximately twice as high EC50 as thiol conjugates (DAR3.3). ..

As shown in FIG. 36, when assayed against SK-BR-3 expressing cells or MDA-MB-231 cells, similar trends carry the A114N hyperglycosylated mutation and AO-MMAE described in Example 14. It was observed in a cell proliferation assay for ADCs prepared with anti-HER antibodies. The A114N complex carbohydrate clearly exhibits enhanced cytotoxicity against Her2-expressing cell lines throughout the non-expressing lineage. Relative toxicity compared to SialT complex carbohydrates prepared with the same antibody is consistent with the lower drug loading of this preparation.

Cell proliferation assays were also performed on ADCs prepared with anti-TEM1 antibodies carrying A114N hyperglycosylated mutations and AO-MMAE prepared as described in Example 14. Higher toxicity was observed for the TEM1-expressing cell lines SJSA-1 and A673 compared to the non-expressing MDA-MB-231 series. The level of toxicity compared to conventional thiol conjugates with the same antibody was consistent with the drug content (DAR) of this preparation.

In summary, site-specific conjugation of drugs with glycans with cleavable linkers to toxic ADCs and conventional thiol-based conjugates, as demonstrated with different antibodies and different drug-linkers. A comparable in vitro effect is produced. Moreover, below 2 mM of periodate, drug conjugate levels correlate with a decrease in sialic acid. As expected from the complete conversion of sialic acid to the oxidized form, increasing the concentration of periodate above 2 mM provides little benefit. However, under all conditions, the number of drugs per antibody is slightly less than the sialic acid content, and some of the oxidized sialic acid is filled or otherwise drug-linker. It is pointed out that it may not be available for coupling as well, either due to steric hindrance in the bulk of the.

In vivo characterization of antibody drug conjugates The efficacy of anti-HER complex carbohydrates was also evaluated in Her2 + tumor cell xenograft mode and compared to thiol conjugates with approximately 2-fold higher DA. bottom. Treatment was started after implanting SK-OV-3 Her2 + tumor cells in Beige / SCID mice and establishing a tumor of approximately 150 mm ³ . ADCs at doses of 3 or 10 mg / kg were injected through the tail vein on days 38, 45, 52, and 59. There were about 10 mice per group. Tumor volumes in different groups of mice were measured and their survival was recorded. Survival curves were plotted based on the Kaplan-Meier method.

FIG. 33 shows a comparison of in vivo efficacy of anti-HER complex carbohydrates and thiol conjugates in a Her2 + tumor cell xenograft model. MMAE (FIGS. 33A and B) and PEG8-Dol10 (FIGS. 33A and B) and PEG8-Dol10 (FIGS. 33A and B), which include a control of a complex carbohydrate or thiol conjugate containing approximately 2-fold higher DAR in Beige / SCID mice implanted with SK-OV-3 Her2 + tumor cells. 33C and D) were administered. The kinetics of tumor growth in MMAE conjugates is shown in FIG. 33A. In this case, the complex carbohydrate was significantly higher than the naked antibody alone (black), but showed lower efficacy than the thiol conjugate comparative control with approximately 2-fold higher DAT (green). MMAE complex carbohydrates showed a significant tumor retraction and a delay of approximately 20 days in tumor growth (FIG. 33A) and a approximately 2-fold increase in survival time from the first dose (FIG. 33B). .. Thiol MMAE conjugates showed almost complete tumor suppression at the same dose of ADC (10 mg / kg).

The in vivo efficacy of the PEG8-Dol10 complex carbohydrate (“GlycoDol10”) and the thiol-conjugated comparative control (“ThiolDol10”) with approximately 2-fold higher DA was also determined in the same Her2 + tumor cell xenograft model. .. Both conjugates showed lower efficacy than the MMAE conjugates described above. However, 10 mg / kg aminooxy-PEG8-Dol10 complex carbohydrate (“GlycoDol10”) showed a 15-day delay in tumor growth (FIG. 33C) and approximately 20 days of survival time after the first dose. It showed an increase of (1.7 times) (Fig. 33D). Thiol conjugates were more effective at the same dose and showed a approximately 2-fold increase in survival. At low doses (3 mg / kg), thiol conjugates showed lower efficacy than 10 mg / kg of complex carbohydrates. This dose corresponds to a dose of 80 μmol of the PEG8-Dol10 drug per kg, whereas for glycoconjugates it is a dose of 110 μmol of the PEG8-Dol10 drug per kg.

These data demonstrate that site-specific conjugation of the antibody glycans onto sialic acid yields molecules with comparable potency to ADCs produced by thiol-based chemistries. .. The somewhat low efficacy of in vivo may be due to the small number of drugs carried into the tumor cells by each antibody due to the internal translocation of the antigen bound to each antibody. We have not compared these complex carbohydrates to thiol conjugates with the same DAR, but observed at various doses of the two ADCs indicating that the administered drug was at comparable levels. The indicated efficacy indicates that the complex carbohydrate has an intrinsic efficacy comparable to its thiol counterpart and points out that there is no adverse effect on the conjugation of this site. In addition, a 10 mg / kg dose of Dol10 complex carbohydrate that introduces only 28% more drug results in a 2-fold increase in survival over thiol conjugates (3 mg / kg), and these conjugates are the same. It is suggested that even DAT can provide excellent efficacy. Higher drug content by a number of different strategies, including the use of branched drug linkers or the introduction of additional glycosylation sites, and using the same method, if there are obvious limitations in the incorporation of sialic acid in natural glycans. Can be achieved.

Claims (35)

At least one equation (IV):
-Gal-Sia-C (H) = NQ-CON-X
Equation (IV)
[During the ceremony,
A) Q is NH or O,
B) CON is a connection part,
C) X is an effector part,
D) Gal is a component derived from galactose and is a component.
E) Sia is a component derived from sialic acid and is a component.
Sia is present or non-existent]
A binding polypeptide comprising at least one modified glycan comprising a moiety. The binding polypeptide according to claim 1, wherein the modified glycan is a bifurcated glycan. The binding polypeptide according to claim 1 or 2, wherein the bifurcated glycan is fucosylated or non-fucosylated. The binding polypeptide according to any one of claims 1 to 3, wherein the modified glycan comprises at least two moieties of formula (IV) and Sia is present in only one of the two moieties. The binding polypeptide according to any one of claims 1 to 3, wherein the modified glycan comprises at least two moieties of formula (IV) and Sia is present in both of the two moieties. The binding polypeptide according to any one of claims 1 to 5, wherein the modified glycan is N-linked to the binding polypeptide. The binding polypeptide according to any one of claims 1 to 6, wherein the binding polypeptide comprises an Fc domain. The binding polypeptide according to claim 7, wherein the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid position 297 of the Fc domain by EU numbering. The binding polypeptide according to claim 8, wherein the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid 298 of the Fc domain by EU numbering. The binding polypeptide according to any one of claims 1 to 9, wherein the Fc domain is human. The binding polypeptide according to any one of claims 1 to 10, wherein the binding polypeptide comprises a CH1 domain. The binding polypeptide according to claim 11, wherein the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid 114 of the CH1 domain by Kabat numbering. The binding polypeptide according to any one of claims 1 to 12, wherein the effector moiety is a cytotoxic agent. The binding polypeptide according to claim 13, wherein the cytotoxicity is selected from the group consisting of the cytotoxicities listed in Table 1. The binding polypeptide according to any one of claims 1 to 13, wherein the effector portion is a detection agent. The binding polypeptide according to any one of claims 1 to 13, wherein the effector moiety is a targeting moiety. The binding polypeptide of claim 16, wherein the targeting moiety is a carbohydrate or glycopeptide. The binding polypeptide of claim 16, wherein the targeting moiety is a glycan. The binding polypeptide of any one of claims 1-18, wherein the connecting moiety comprises a pH sensitive linker, a disulfide linker, an enzyme sensitive linker, or another cleavable linker moiety. The binding polypeptide according to any one of claims 1 to 19, wherein the connecting moiety comprises a linker moiety selected from the group of linker moieties shown in Table 2 or 14. The binding polypeptide according to any one of claims 1 to 20, which is an antibody or immunoadhesin. A composition comprising the binding polypeptide according to any one of claims 1 to 21 and a pharmaceutically acceptable carrier or excipient. 22. The composition of claim 22, wherein the therapeutic or diagnostic effector moiety has a ratio of less than 4 to the binding polypeptide. 23. The composition of claim 23, wherein the therapeutic or diagnostic effector moiety has a ratio of about 2 to the binding polypeptide. A method of treating a patient in need thereof comprising administering an effective amount of the composition of claim 24. An isolated polynucleotide encoding the binding polypeptide according to any one of claims 1-21. A vector comprising the polynucleotide according to claim 26. A host cell comprising the polynucleotide or vector according to claim 26 or 27. The method for producing the binding polypeptide according to any one of claims 1 to 28, wherein the formula (I):
NH ₂ -Q-CON-X
Equation (I)
[During the ceremony,
A) Q is NH or O,
B) CON is a connection part,
C) X is the effector part]
The method comprising reacting an effector moiety of the same with a modified binding polypeptide containing an oxidized glycan. 29. The method of claim 29, wherein the modified binding polypeptide containing the oxidized glycan is produced by reacting the binding polypeptide containing the glycan with a mild oxidizing agent. 30. The method of claim 30, wherein the mild oxidant is sodium periodate. 31. The method of claim 31, wherein less than 1 mM sodium periodate is used. 30. The method of claim 30, wherein the oxidizing agent is galactose oxidase. The method of any one of claims 29-33, wherein the binding polypeptide comprising a glycan comprises one or two terminal sialic acid residues. 34. The method of claim 34, wherein the terminal sialic acid residue is introduced by treating the binding polypeptide with sialyltransferase, or a combination of sialyltransferase and galactosyltransferase.

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